Treatment of atopic dermatitis and asthma using RNA complexes that target IL4Rα, TRPA1, or F2RL1

ABSTRACT

In certain aspects, provided herein are RNA complexes (e.g., asymmetric RNA complexes, such as asiRNAs or cell penetrating asiRNAs) that inhibit IL4Rα, TRPA1, and/or F2RL1 expression and are therefore useful for treating atopic dermatitis or asthma.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/290,298, filed Feb. 2, 2016, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 10, 2017, is named OPH-00701_SL.txt and is 176,290 bytes in size.

BACKGROUND

Dysregulation of the immune system can result in autoimmune diseases such as atopic dermatitis and asthma. Atopic dermatitis, also referred to as eczema, is an inflammatory disease characterized by the presence of itchy and tender skin, edema, and erythema. Atopic dermatitis is common in children and infants, although the disease can occur at any age.

About 70% of atopic dermatitis patients develop asthma by “atopic march,” characterized by the progression of atopic dermatitis to asthma and allergic rhinitis. Asthma is a respiratory disorder also associated with dysregulation of the immune system. More specifically, it is a chronic respiratory disease marked by respiratory spasms and obstruction due to allergic inflammation of the bronchi, causing repetitive breathing shortness, wheezing and coughing. Asthma prevalence is estimated to be as high as 300 million individuals worldwide, and about 8% of the population of major developed countries are afflicted with asthma.

IL4Rα, F2RL1 and TRPA1 genes play a key role in the onset and progression of symptoms of atopic dermatitis and/or asthma. When exposed to foreign antigens, dendritic cells in atopic dermatitis patients activate Th2 cells, leading to the secretion of cytokines (e.g., IL-4, IL-5, IL-10, and IL-13) by the activated Th2 cells. Among the cytokines, IL-4 and IL-13 are known to play an important role in the onset of atopic dermatitis, while IL-4 and IL-13 have been reported to worsen atopic dermatitis symptoms of through the inhibition of human beta defensin-3 and filaggrin, both of which maintain the skin barrier. The receptors for IL-4 and IL-13 are heterodimers and contain IL4Rα (interleukin 4 receptor, alpha, also known as IL4Rα). Therefore, down-regulation of the IL4Rα can block out the signals of IL-4 and IL-13.

The main cause of the itching symptom experienced by atopic dermatitis patients is the overexpression of thymic stromal lymphopoietin (TSLP) in keratinocytes, which elevates the transient receptor potential (TRP) of TRP ion channels, including TRPV1 and TRPA1. Thus, the symptoms of atopic dermatitis can be treated by the inhibition of TRPA1.

Coagulation factor II (thrombin) receptor-like 1 (F2RL1, also known as protease-activated receptor 2, PAR2) is expressed by keratinocytes, activated endothelial cells, and sensory nerves in the skin and is involved in various inflammation reactions, pigmentation production, and the skin barrier function. F2RL1 plays a pivotal role in the activation of proteinases, which induce inflammation reactions and the aggravated skin conditions seen in atopic dermatitis patients.

Thus, there is a need for new and improved therapeutics targeting IL4Rα, TRPA1 and F2RL1 for the treatment or atopic dermatitis or asthma.

SUMMARY

In certain aspects, provided herein are RNA complexes that target IL4Rα, TRPA1, or F2RL1 and are useful for treating and/or preventing atopic dermatitis and/or asthma. In certain aspects, provided herein are pharmaceutical compositions comprising such RNA complexes and methods of using such RNA complexes and pharmaceutical compositions.

In certain aspects, provided herein is an RNA complex comprising an antisense strand having sequence complementarity to an IL4Rα, TRPA1, or F2RL1 mRNA sequence and a sense strand having sequence complementarity to the antisense strand. In some embodiments, the RNA complex is capable of inhibiting IL4Rα, TRPA1, or F2RL1 expression by a cell (e.g., a keratinocyte). In some embodiments, the RNA complex is an asymmetric shorter-duplex small interfering RNA (an asiRNA). In some embodiments, the RNA complex is an RNA complex listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, or Table 10. In some embodiments, the RNA complex comprises the antisense and sense strand of IL4RA#5. In some embodiments, the RNA complex comprises the antisense and sense strand of TRPA1#81. In some embodiments, the RNA complex comprises the antisense and sense strand of F2RL1#22.

In some embodiments, the RNA complex provided herein comprises a chemical modification, wherein the modification facilitates the penetration of a cellular membrane in the absence of a delivery vehicle. In some embodiments, the modification is a 2′-O-methylated nucleoside, a phosphorothioate bond or a hydrophobic moiety. In some embodiments, the RNA complexes provided herein comprise a hydrophobic moiety. In some embodiments, the hydrophobic moiety can be any chemical structure having hydrophobic character. For example, in some embodiments the hydrophobic moiety is a lipid, a lipophilic peptide and/or a lipophilic protein. In some embodiments, the hydrophobic moiety is a lipid, such as cholesterol, tocopherol, or a long-chain fatty acid having 10 or more carbon atoms (e.g., stearic acid or palmitic acid). In some embodiments, the hydrophobic moiety is cholesterol. In some embodiments, the RNA complex is a modified RNA complex listed in Table 2, Table 3, Table 5, Table 6, Table 8, Table 9, or Table 10. In certain embodiments, the RNA complex is not cytotoxic.

In certain aspects, provided herein is a pharmaceutical composition comprising an RNA complex provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is formulated for topical delivery. In some embodiments, the pharmaceutical composition is a cream or a lotion. In some embodiments, the pharmaceutical composition is formulated for parenteral, intravenous, or oral delivery. In other embodiments, the pharmaceutical composition is formulated for inhalation.

In certain aspects, provided herein is a method of inhibiting IL4Rα, TRPA1, or F2RL1 expression by a cell, comprising contacting the cell with an RNA complex provided herein.

In certain aspects, provided herein is a method of inhibiting gene expression IL4Rα, TRPA1, or F2RL1 in a human subject comprising administering to the subject an RNA complex or pharmaceutical composition provided herein. In certain aspects, provided herein is a method of treating a human subject for atopic dermatitis and/or asthma comprising administering to the subject an RNA complex or pharmaceutical composition provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene silencing efficiency of 73 exemplary asiRNAs that target IL4Rα.

FIG. 2 shows the gene silencing efficiency of 15 exemplary asiRNAs that target IL4Rα.

FIG. 3 shows the gene silencing effects of 2 exemplary asiRNAs that target IL4Rα.

FIG. 4 shows the gene silencing efficiency of exemplary IL4Rα-targeting cell-penetrating asiRNAs (IL4Rα cp-asiRNAs) to which various chemical modifications have been applied.

FIG. 5 shows the inhibition of IL4Rα protein expression by exemplary cp-asiRNAs.

FIG. 6 shows the gene silencing efficiency of 4 cp-asiRNAs of different antisense strand lengths (19 or 21 nucleotides).

FIG. 7 shows the inhibition of IL4Rα protein expression by 4 exemplary cp-asiRNAs.

FIG. 8 provides the human IL4Rα mRNA sequence. Figure discloses SEQ ID NO: 678.

FIG. 9 shows the gene silencing efficiency of 102 exemplary asiRNAs that target TRPA1.

FIG. 10 shows the gene silencing effects of 14 exemplary asiRNAs that target TRPA1.

FIG. 11 shows the inhibition of TRPA1 protein expression by 14 exemplary asiRNAs that target TRPA1.

FIG. 12 shows the gene silencing efficiency of exemplary TRPA1-targeting cell-penetrating asiRNAs (TRPA1 cp-asiRNAs) to which various chemical modifications have been applied.

FIG. 13 shows the inhibition of TRPA1 protein expression by exemplary cp-asiRNAs.

FIG. 14 shows the gene silencing efficiency of 8 cp-asiRNAs of different antisense strand lengths (19 or 21 nucleotides) and different sense strand chemical modifications (3 or 4 phosphorothioate bond).

FIG. 15 shows the inhibition of TRPA1 protein expression by 8 exemplary cp-asiRNAs.

FIG. 16 shows the inhibition of TRPA1 protein expression by 4 exemplary cp-asiRNAs. A549 cells were incubated with 1 μM and 3 μM cp-asiRNAs in the absence of transfection reagent.

FIG. 17 provides the human TRPA1 mRNA sequence. Figure discloses SEQ ID NO: 679.

FIG. 18 shows the gene silencing efficiency of 100 exemplary asiRNAs that target F2RL1.

FIG. 19 shows the gene silencing efficiency of 29 exemplary asiRNAs that target F2RL1.

FIG. 20 shows the gene silencing efficiency of 32 exemplary asiRNAs containing 2′-O-Methylation modifications.

FIG. 21 shows the gene silencing effects of 12 exemplary asiRNAs that target F2RL1.

FIG. 22 shows the inhibition of F2RL1 protein expression by 12 exemplary asiRNAs that target F2RL1.

FIG. 23 shows the gene silencing efficiency of exemplary F2RL1-targeting cell-penetrating asiRNAs (cp-asiRNAs or cp-asiF2RL1s) to which various chemical modifications have been applied.

FIG. 24 shows the inhibition of F2RL1 mRNA expression by exemplary cp-asiRNAs.

FIG. 25 shows the inhibition of F2RL1 protein expression by exemplary cp-asiRNAs.

FIG. 26 shows the gene silencing efficiency of 8 cp-asiRNAs of different antisense strand lengths (19 or 21 nucleotides).

FIG. 27 shows the inhibition of F2RL1 protein expression by 8 exemplary cp-asiRNAs.

FIG. 28 shows the mRNA sequence of human F2RL1. Figure discloses SEQ ID NO: 680.

FIG. 29 shows the experimental timeline of cp-asiRNA treatment in an induced model of atopic dermatitis.

FIG. 30 shows scratching time observed in Dermatophagoides farinae body extract (Df) cream treated samples.

FIG. 31 shows intradermal injection versus cream cp-asiRNA application in rodents of atopic dermatitis.

FIG. 32 shows H&E staining of skin sections and quantified epidermis area by analyzing skin section image of a rodent model of atopic dermatitis.

FIG. 33 shows mast cell infiltration analysis of treated skin region.

DETAILED DESCRIPTION General

In certain aspects, provided herein are asymmetric RNA complexes (e.g., asiRNAs or cp-asiRNAs) that inhibit IL4Rα, TRPA1, and/or F2RL1 and are therefore useful for the treatment of atopic dermatitis and/or asthma. In some embodiments, the RNA complexes are chemically modified to be capable of penetrating a cell without need for a transfection vehicle. In some embodiments, the RNA complex is an RNA complex listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, or Table 10. In certain aspects, provided herein are pharmaceutical compositions comprising such RNA complexes and methods of using such RNA complexes and pharmaceutical compositions.

In some embodiments, the RNA complexes described herein are asiRNAs or cp-asiRNAs. As used herein, the term asiRNA refers to double-stranded asymmetrical short interfering RNA molecules that have a 19-21 nt antisense strand and a 13-17 nt sense strand. Additional information on asiRNAs can be found in U.S. Pat. Pub. No. 2012/0238017 and in Chang et al., Mol. Ther. 17:725-732 (2009), each of which is hereby incorporated by reference in its entirety.

In some embodiments, the RNA complexes described herein are delivered to cells using a delivery vehicle, such as liposomes, cationic polymers, cell penetrating peptides (CPPs), protein transduction domains (PTDs), antibodies and/or aptamers. In some embodiments, the RNA complex described herein is chemically modified so as to not require the use of such delivery vehicles to mediate IL4Rα, TRPA1, and/or F2RL1 inhibition in a cell. Such RNA complexes are referred to herein as cell-penetrating asiRNAs (cp-asiRNAs).

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein, the term “administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

As used herein, the term “immunomodulator” refers to a compound or composition which weakens, stimulate, or otherwise modulates the immune system. Examples include, by are not limited to leukotriene receptor agonists, immunosuppressants (e.g., FK-506), or cytokines.

As used herein, the terms “interfering nucleic acid” and “inhibiting nucleic acid” are used interchangeably. Interfering nucleic acids generally include a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically RNA) by Watson-Crick base pairing, to form a nucleic acid: oligomer heteroduplex within the target sequence. Interfering RNA molecules include, but are not limited to, antisense molecules, siRNA molecules, asiRNA molecules, cp-asiRNA molecules, single-stranded siRNA molecules, miRNA molecules and shRNA molecules. Such an interfering nucleic acids can be designed to block or inhibit translation of mRNA or to inhibit natural pre-mRNA splice processing, or induce degradation of targeted mRNAs, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes. Interfering nucleic acids may include, for example, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), 2′-0-Methyl oligonucleotides and RNA interference agents (siRNA agents). RNAi molecules generally act by forming a heteroduplex with the target molecule, which is selectively degraded or “knocked down,” hence inactivating the target RNA. Under some conditions, an interfering RNA molecule can also inactivate a target transcript by repressing transcript translation and/or inhibiting transcription of the transcript. An interfering nucleic acid is more generally said to be “targeted against” a biologically relevant target, such as a protein, when it is targeted against the nucleic acid of the target in the manner described above.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides, whether deoxyribonucleotides, ribonucleotides, or analogs thereof, in any combination and of any length. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, Unucleobases are interchangeable with T nucleobases.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material.

An oligonucleotide “specifically hybridizes” to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions, with a Tm substantially greater than 45° C., or at least 50° C., or at least 60° C.-80° C. or higher. Such hybridization corresponds to stringent hybridization conditions. At a given ionic strength and pH, the Tm is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Again, such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity.

As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy.

The phrases “therapeutically-effective amount” and “effective amount” as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment.

“Treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

As used herein, a therapeutic that “prevents” a disorder or condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

RNA Complexes

In certain aspects, provided herein are RNA complexes that target IL4Rα, TRPA1, and/or F2RL1 mRNA and inhibit IL4Rα, TRPA1, and/or F2RL1 expression by a cell, respectively. The nucleic acid sequence of human IL4Rα, TRPA1, and F2RL1 mRNA is provided in FIG. 8, FIG. 17, and FIG. 28, respectively.

In certain aspects, provided herein is an RNA complex comprising an antisense strand having sequence complementarity to an IL4Rα, TRPA1, or F2RL1 mRNA sequence (e.g., a human IL4Rα, TRPA1, or F2RL1 mRNA sequence) and a sense strand having sequence complementarity to the antisense strand. In some embodiments, the RNA complex is capable of inhibiting IL4Rα, TRPA1, or F2RL1 expression by a cell. In some embodiments, the RNA complex is an asymmetric shorter-duplex small interfering RNA (an asiRNA). In some embodiments, the RNA complex is an RNA complex listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 8 or Table 10. The RNA complexes described herein can contain RNA bases, non-RNA bases or a mixture of RNA bases and non-RNA bases. For example, certain RNA complexes provided herein can be primarily composed of RNA bases but also contain DNA bases or non-naturally occurring nucleotides.

In some embodiments, the antisense strand is at least 19 nucleotides (nt) in length. In some embodiments, the antisense strand is 19 to 21 nt in length (i.e., 19, 20 or 21 nt in length). In some embodiments, at least 13, 14, 15, 16, 17, 18, 19, 20 or 21 nt of the antisense strand are complementary to the IL4Rα, TRPA1, or F2RL1 mRNA sequence. Perfect complementarity is not necessary. In some embodiments, the antisense strand is perfectly complementary to the IL4Rα, TRPA1, or F2RL1 mRNA sequence.

In some embodiments, the antisense strand is at least 24 nt in length (e.g., at least 25 nt in length, at least 26 nt in length, at least 27 nt in length, at least 28 nt in length, at least 29 nt in length, at least 30 nt in length or at least 31 nt in length). In some embodiments, the antisense strand is no greater than 124 nt in length (e.g., no greater than 100 nt in length, no greater than 90 nt in length, no greater than 80 nt in length, no greater than 70 nt in length, no greater than 60 nt in length, no greater than 50 nt in length or no greater than 40 nt in length. In some embodiments, the antisense strand is 31 nt in length. In some embodiments, at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, 30 or 31 nt of the antisense strand are complementary to the IL4Rα, TRPA1, or F2RL1 mRNA sequence. Perfect complementarity is not necessary. In some embodiments, the antisense strand is perfectly complementary to the IL4Rα, TRPA1, or F2RL1 mRNA sequence.

In some embodiments, the sense strand is 15 to 17 nt in length (i.e., 15 nt in length, 16 nt in length or 17 nt in length). In some embodiments, at least 15 nt, at least 16 nt or at least 17 nt of the sense strand are complementary to the sequence of the antisense strand. In some embodiments the sense strand is perfectly complementary to the sequence of the antisense strand.

In some embodiments, the antisense strand and the sense strand form a complex in which the 5′ end of the antisense strand and the 3′ end of the sense strand form a blunt end. In some embodiments, the antisense strand and the sense strand form a complex in which the 5′ end of the antisense strand overhangs the 3′ end of the sense strand (e.g., by 1, 2, 3, 4 or 5 nt). In some embodiments, the antisense strand and the sense strand form a complex in which the 5′ end of the sense strand overhangs the 3′ end of the antisense strand (e.g., by 1, 2, 3, 4 or 5 nt).

In some embodiments, the antisense strand and/or the sense strand of the RNA complex has a sense strand sequence and/or an antisense strand sequence selected from the sequences listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 8 or Table 10.

In some embodiments, the RNA complex provided herein comprises a chemical modification, wherein the modification facilitates the penetration of a cellular membrane in the absence of a delivery vehicle. In some embodiments, the modification is a 2′-O-methylated nucleoside, a phosphorothioate bond or a hydrophobic moiety. In some embodiments, the chemical modification is a hydrophobic moiety. In some embodiments, the hydrophobic moiety is a cholesterol moiety. In some embodiments, the RNA complex is a modified RNA complex listed in Table 2, table 3, Table 5, Table 6, Table 8, Table 9, or Table 10. In certain embodiments, the RNA complex is not cytotoxic.

The RNA complexes described herein can employ a variety of oligonucleotide chemistries. Examples of oligonucleotide chemistries include, without limitation, peptide nucleic acid (PNA), linked nucleic acid (LNA), phosphorothioate, 2′O-Me-modified oligonucleotides, and morpholino chemistries, including combinations of any of the foregoing. In general, PNA chemistries can utilize shorter targeting sequences because of their relatively high target binding strength relative to 2′O-Me oligonucleotides. Phosphorothioate and 2′O-Me-modified chemistries are often combined to generate 2′O-Me-modified oligonucleotides having a phosphorothioate backbone. See, e.g., PCT Publication Nos. WO/2013/112053 and WO/2009/008725, each of which is hereby incorporated by reference in its entirety.

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition. The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications (see structure below). The backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.

Despite a radical structural change to the natural structure, PNAs are capable of sequence-specific binding in a helix form to DNA or RNA. Characteristics of PNAs include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA. PANAGENE™ has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2-sulfonyl group) and proprietary oligomerization process. The PNA oligomerization using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping. PNAs can be produced synthetically using any technique known in the art. See, e.g., U.S. Pat. Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006 and 7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teaching of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991. Each of the foregoing is incorporated by reference in its entirety.

Interfering nucleic acids may also contain “locked nucleic acid” subunits (LNAs). “LNAs” are a member of a class of modifications called bridged nucleic acid (BNA). BNA is characterized by a covalent linkage that locks the conformation of the ribose ring in a C3-endo (northern) sugar pucker. For LNA, the bridge is composed of a methylene between the 2′-O and the 4′-C positions. LNA enhances backbone preorganization and base stacking to increase hybridization and thermal stability.

The structures of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, and Accounts of Chem. Research (1999) 32:301); Obika, et al., Tetrahedron Letters (1997) 38:8735; (1998) 39:5401, and Bioorganic Medicinal Chemistry (2008) 16:9230. Compounds provided herein may incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs. Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are described, for example, in U.S. Pat. Nos. 7,572,582, 7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and 6,670,461, each of which is incorporated by reference in its entirety. Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed. One embodiment is an LNA-containing compound where each LNA subunit is separated by a DNA subunit. Certain compounds are composed of alternating LNA and DNA subunits where the intersubunit linker is phosphorothioate.

In certain embodiments, the RNA complex is linked to a cholesterol moiety. In some embodiments, the cholesterol moiety is attached to the 3′ terminus of the sense strand. In some embodiments, the cholesterol moiety is attached to the 3′ terminus of the antisense strand. In some embodiments, the cholesterol moiety is attached to the 5′ terminus of the sense strand. In some embodiments, the cholesterol moiety is attached to the 5′ terminus of the antisense strand.

In some embodiments, the RNA complex comprises a 2′-O-methylated nucleoside. 2′-O-methylated nucleosides carry a methyl group at the 2′-OH residue of the ribose molecule. 2′-O-Me-RNAs show the same (or similar) behavior as RNA, but are protected against nuclease degradation. 2′-O-Me-RNAs can also be combined with phosphothioate oligonucleotides (PTOs) for further stabilization. 2′-O-Me-RNAs (phosphodiester or phosphothioate) can be synthesized according to routine techniques in the art (see, e.g., Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is hereby incorporated by reference).

In some embodiments, the 2′-O-methyl nucleoside is positioned at the 3′ terminus of the sense strand. In some embodiments, 3′ terminal region of the sense strand comprises a plurality of 2′-O-methylated nucleosides (e.g., 2, 3, 4, 5 or 6 2′-O-methylated nucleosides within 6 nucleosides of the 3′ terminus). In some embodiments, the 2′-O-methyl nucleoside is positioned at the 3′ terminus of the antisense strand. In some embodiments, 3′ terminal region of the antisense strand comprises a plurality of 2′-O-methylated nucleosides (e.g., 2, 3, 4, 5 or 6 2′-O-methylated nucleosides within 6 nucleosides of the 3′ terminus). In some embodiments, both the 3′ terminal region of the sense strand and the 3′ terminal region of the antisense strand comprise a plurality of 2′-O-methylated nucleosides. In some embodiments, the sense strand comprises 2′-O-methylated nucleosides that alternate with unmodified nucleosides. In some embodiments, the sense strand comprises a contiguous sequence of 2, 3, 4, 5, 6, 7 or 8 2′-O-methylated nucleosides that alternate with unmodified nucleosides. In some embodiments, the anti-sense strand comprises 2′-O-methylated nucleosides that alternate with unmodified nucleosides. In some embodiments, the anti-sense strand comprises a contiguous sequence of 2, 3, 4, 5, 6, 7 or 8 2′-O-methylated nucleosides that alternate with unmodified nucleosides.

In some embodiments, the RNA complex comprises a phosphorothioate bond. “Phosphorothioates” (or S-oligos) are a variant of normal DNA in which one of the non-bridging oxygens is replaced by a sulfur. The sulfurization of the internucleotide bond reduces the action of endo- and exonucleases including 5′ to 3′ and 3′ to 5′ DNA POL 1 exonuclease, nucleases 51 and P1, RNases, serum nucleases and snake venom phosphodiesterase. Phosphorothioates are made by two principal routes: by the action of a solution of elemental sulfur in carbon disulfide on a hydrogen phosphonate, or by the method of sulfurizing phosphite triesters with either tetraethylthiuram disulfide (TETD) or 3H-1,2-benzodithiol-3-one 1,1-dioxide (BDTD) (see, e.g., Iyer et al., J. Org. Chem. 55, 4693-4699, 1990). The latter methods avoid the problem of elemental sulfur's insolubility in most organic solvents and the toxicity of carbon disulfide. The TETD and BDTD methods also yield higher purity phosphorothioates.

In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the bonds between the ribonucleotides in the sense strand of the RNA complex are phosphorothioate bonds. In some embodiments, all of the bonds between the ribonucleotides in the sense strand of the RNA complex are phosphorothioate bonds.

In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the bonds between the ribonucleotides in the antisense strand of the RNA complex are phosphorothioate bonds. In some embodiments, all of the bonds between the ribonucleotides in the antisense strand of the RNA complex are phosphorothioate bonds.

The RNA complexes described herein may be contacted with a cell or administered to an organism (e.g., a human). Alternatively, constructs and/or vectors encoding the RNA complexes may be contacted with or introduced into a cell or organism. In certain embodiments, a viral, retroviral or lentiviral vector is used.

The RNA complexes described herein can be prepared by any appropriate method known in the art. For example, in some embodiments, the RNA complexes described herein are prepared by chemical synthesis or in vitro transcription.

In certain aspects, provided herein is a pharmaceutical composition comprising an RNA complex provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is formulated for delivery to the skin (e.g., as a cream or lotion). In certain embodiments, the pharmaceutical composition is formulated for delivery to the lungs (e.g., as an inhaler). In some embodiments, the pharmaceutical composition is formulated for oral or parenteral delivery. In some embodiments, the pharmaceutical composition further comprises a second agent for treatment of atopic dermatitis or asthma. In some embodiments, the second agent is a steroid (e.g., a corticosteroid), a long acting beta agonist (e.g., salmenterol or formoterol), or an immunomodulator. Examples of steroids include hydrocortisone, fluticasone, mudesonide, mometasone, beclomethasone, ciclesonide or flunisolide. Examples of immunomodulators include montelukast, zafirlukast, or zileuton. Two or more steroids, long acting beta agonists, and immunomodulators may be taken in with the pharmaceutical composition.

In some embodiments, the pharmaceutical composition is formulated for delivery to the skin. In some embodiments, the composition is an emulsion, cream, lotion, gel, oil, ointment, aerosol spray, or semi-solid formulation. In some embodiments, the topical formulation comprises a carrier selected from trehalose, malto-dextrin, rice flour, micro-crystalline cellulose, magnesium stearate, inositol, fructo-oligosaccharide, gluco-oligosaccharide, dextrose, sucrose, talc, water, physiological salt solution, urea, methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, white pertrolatum, isopropyl myristate, lanolin, lanolin alcohol, mineral oil, lavender oil, nasturtium extract oil, sorbitan mono-oleate, cetylstearyl alcohol, hydroxypropyl cellulose, detergent, sucrose stearate, sucrose cocoate, sucrose distearate, 2-ethyl-1,3-hexanediol, polyoxypropylene-15-stearyl ether, glycerol stearate, glycerin, synthetic spermaceti, cetyl alcohol, butylparaben, propylparaben, and methylparaben.

In certain embodiments, the pharmaceutical composition does not comprise a transfection vehicle. In some embodiments, the pharmaceutical composition comprises a delivery vehicle (e.g., liposomes, cationic polymers, cell penetrating peptides (CPPs), protein transduction domains (PTDs), antibodies and/or aptamers). In some embodiments, the composition includes a combination of multiple (e.g., two or more) of the RNA complexes described herein.

Methods of preparing these formulations or compositions include the step of bringing into association an RNA complex described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers.

Therapeutic Methods

In certain aspects, provided herein is a method of inhibiting IL4Rα, TRPA1, or F2RL1 expression by a cell, comprising contacting the cell with an RNA complex provided herein. In some embodiments, the RNA complex is a modified RNA complex and the cell is contacted with the RNA complex in the absence of a transfection vehicle. In some embodiments, the cell is contacted with the RNA complex in the presence of a delivery vehicle (e.g., a liposome, cationic polymer, cell penetrating peptide (CPP), protein transduction domain (PTD), antibody and/or aptamer). In some embodiments, the cell is present in the respiratory tract of a human subject. In some embodiments, the cell is present in the skin of a human subject. In some embodiments, the subject has atopic dermatitis. In some embodiments, the subject has asthma. In some embodiments, the subject is female. In some embodiments, the subject is male.

In certain aspects, provided herein is a method of treating a human subject for atopic dermatitis and/or asthma comprising administering to the subject an RNA complex or pharmaceutical composition provided herein. In certain embodiments, the RNA complex or pharmaceutical composition is administered to the respiratory tract of the subject. In certain embodiments, the RNA complex or pharmaceutical composition is administered to the skin of the subject. In some embodiments, the RNA complex or pharmaceutical composition self-administered by the subject.

In the present methods, an RNA complex described herein can be administered to the subject, for example, as nucleic acid without delivery vehicle (e.g., for cp-asiRNAs), in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express the RNA complex described herein. In some embodiments, any nucleic acid delivery method known in the art can be used in the methods described herein. Suitable delivery reagents include, but are not limited to, e.g., the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules is described in Minakuchi et al. Nucleic Acids Res., 32(13):e109 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008); each of which is incorporated herein in their entirety. Exemplary interfering nucleic acid delivery systems are provided in U.S. Pat. Nos. 8,283,461, 8,313,772, 8,501,930. 8,426,554, 8,268,798 and 8,324,366, each of which is hereby incorporated by reference in its entirety.

In some embodiments of the methods described herein, liposomes are used to deliver an RNA complex described herein to a subject. Liposomes suitable for use in the methods described herein can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng. 9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which are herein incorporated by reference.

The liposomes for use in the present methods can also be modified so as to avoid clearance by the mononuclear macrophage system (“MMS”) and reticuloendothelial system (“RES”). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure.

Opsonization-inhibiting moieties for use in preparing the liposomes described herein are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is “bound” to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer that significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference.

In some embodiments, opsonization inhibiting moieties suitable for modifying liposomes are water-soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, or from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. In some embodiments, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called “PEGylated liposomes.”

The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including topically, through inhalation, orally, and parenterally. In certain embodiments, the pharmaceutical compositions are delivered systemically (e.g., via oral or parenteral administration). In certain other embodiments, the pharmaceutical compositions are delivered locally through inhalation into the lungs or topically onto the skin. In some embodiments, the pharmaceutical composition is administered via intradermal injection.

Actual dosage levels of the RNA complexes in the pharmaceutical compositions may be varied so as to obtain an amount of RNA complex that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the agents employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of an RNA complex described herein will be that amount of the RNA complex which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

EXEMPLIFICATION Example 1: Screening for IL4Rα-Specific Asymmetric Shorter-Duplex Small Interfering RNAs

To identify asymmetric shorter-duplex small interfering RNAs (asiRNAs) that inhibit IL4Rα with high efficiency, 73 asiRNAs were synthesized and screened. The nucleic acid sequences of the screened asiRNAs are provided in Table 1.

TABLE 1 Nucleic acid sequences for exemplary IL4Rα-targeting asiRNA. SEQ ID NO: SEQUENCE   1 IL4Rα #1(S): 5′ AUCACCAAGAUUAAGA 3′   2 IL4Rα #1(AS): 5′ UCUUAAUCUUGGUGAUGCUGA 3′   3 IL4Rα #2(S): 5′ UCACCAAGAUUAAGAA 3′   4 IL4Rα #2(AS): 5′ UUCUUAAUCUUGGUGAUGCUG 3′   5 IL4Rα #3(S): 5′ GCCUUCUCAAGCCUGC 3′   6 IL4Rα #3(AS): 5′ GCAGGCUUGAGAAGGCCUUGU 3′   7 IL4Rα #4(S): 5′ CCUUCUCAAGCCUGCU 3′   8 IL4Rα #4(AS): 5′ AGCAGGCUUGAGAAGGCCUUG 3′   9 IL4Rα #5(S): 5′ UGCGUCUCCGACUACA 3′  10 IL4Rα #5(AS): 5′ UGUAGUCGGAGACGCAGGUGG 3′  11 IL4Rα #6(S): 5′ GCGUCUCCGACUACAU 3′  12 IL4Rα #6(AS): 5′ AUGUAGUCGGAGACGCAGGUG 3′  13 IL4Rα #7(S): 5′ GUGGAAGGGCUCCUUC 3′  14 IL4Rα #7(AS): 5′ GAAGGAGCCCUUCCACAGCAG 3′  15 IL4Rα #8(S) 5′ UGGAAGGGCUCCUUCA 3′  16 IL4Rα #8(AS): 5′ UGAAGGAGCCCUUCCACAGCA 3′  17 IL4Rα #9(S): 5′ CAUCACCAAGAUUAAG 3′  18 IL4Rα #9(AS): 5′ CUUAAUCUUGGUGAUGCUGAC 3′  19 IL4Rα #10(S): 5′ CACCAAGAUUAAGAAA 3′  20 IL4Rα #10(AS): 5′ UUUCUUAAUCUUGGUGAUGCU 3′  21 IL4Rα #11(S): 5′ UGGGAUCAGAUUCCCA 3′  22 IL4Rα #11(AS): 5′ UGGGAAUCUGAUCCCACCAUU 3′  23 IL4Rα #12(S): 5′ GGGAUCAGAUUCCCAA 3′  24 IL4Rα #12(AS): 5′ UUGGGAAUCUGAUCCCACCAU 3′  25 IL4Rα #13(S): 5′ AAGACAGUCCUCUGGC 3′  26 IL4Rα #13(AS): 5′ GCCAGAGGACUGUCUUGCUGA 3′  27 IL4Rα #14(S): 5′ AGACAGUCCUCUGGCC 3′  28 IL4Rα #14(AS): 5′ GGCCAGAGGACUGUCUUGCUG 3′  29 IL4Rα #15(S): 5′ GACAGUCCUCUGGCCA 3′  30 IL4Rα #15(AS): 5′ UGGCCAGAGGACUGUCUUGCU 3′  31 IL4Rα #16(S): 5′ ACAGUCCUCUGGCCAG 3′  32 IL4Rα #16(AS): 5′ CUGGCCAGAGGACUGUCUUGC 3′  33 IL4Rα #17(S): 5′ CAGUCCUCUGGCCAGA 3′  34 IL4Rα #17(AS): 5′ UCUGGCCAGAGGACUGUCUUG 3′  35 IL4Rα #18(S): 5′ AGUCCUCUGGCCAGAG 3′  36 IL4Rα #18(AS): 5′ CUCUGGCCAGAGGACUGUCUU 3′  37 IL4Rα #19(S): 5′ GUCCUCUGGCCAGAGA 3′  38 IL4Rα #19(AS): 5′ UCUCUGGCCAGAGGACUGUCU 3′  39 IL4Rα #20(S): 5′ CUCCAGCAUGGGGCAG 3′  40 IL4Rα #20(AS): 5′ CUGCCCCAUGCUGGAGGACAU 3′  41 IL4Rα #21(S): 5′ GGCUAUCAGGAGUUUG 3′  42 IL4Rα #21(AS): 5′ CAAACUCCUGAUAGCCACUGG 3′  43 IL4Rα #22(S): 5′ GCUAUCAGGAGUUUGU 3′  44 IL4Rα #22(AS): 5′ ACAAACUCCUGAUAGCCACUG 3′  45 IL4Rα #23(S): 5′ CUUCUCAAGCCUGCUU 3′  46 IL4Rα #23(AS): 5′ AAGCAGGCUUGAGAAGGCCUU 3′  47 IL4Rα #24(S): 5′ AAUGGGGUGGCUUUGC 3′  48 IL4Rα #24(AS): 5′ GCAAAGCCACCCCAUUGGGAG 3′  49 IL4Rα #25(S): 5′ AUGGGGUGGCUUUGCU 3′  50 IL4Rα #25(AS): 5′ AGCAAAGCCACCCCAUUGGGA 3′  51 IL4Rα #26(S): 5′ CGUCUCCGACUACAUG 3′  52 IL4Rα #26(AS): 5′ CAUGUAGUCGGAGACGCAGGU 3′  53 IL4Rα #27(S): 5′ GACAGUUCACACCAAU 3′  54 IL4Rα #27(AS): 5′ AUUGGUGUGAACUGUCAGGUU 3′  55 IL4Rα #28(S): 5′ ACAGUUCACACCAAUG 3′  56 IL4Rα #28(AS): 5′ CAUUGGUGUGAACUGUCAGGU 3′  57 IL4Rα #29(S): 5′ CAGUUCACACCAAUGU 3′  58 IL4Rα #29(AS): 5′ ACAUUGGUGUGAACUGUCAGG 3′  59 IL4Rα #30(S): 5′ AGUUCACACCAAUGUC 3′  60 IL4Rα #30(AS): 5′ GACAUUGGUGUGAACUGUCAG 3′  61 IL4Rα #31(S): 5′ CUGGAGUGAGUGGAGC 3′  62 IL4Rα #31(AS): 5′ GCUCCACUCACUCCAGGUGGU 3′  63 IL4Rα #32(S): 5′ CAGCAUCACCAAGAUU 3′  64 IL4Rα #32(AS): 5′ AAUCUUGGUGAUGCUGACAUA 3′  65 IL4Rα #33(S): 5′ AGCAUCACCAAGAUUA 3′  66 IL4Rα #33(AS): 5′ UAAUCUUGGUGAUGCUGACAU 3′  67 IL4Rα #34(S): 5′ GCAUCACCAAGAUUAA 3′  68 IL4Rα #34(AS): 5′ UUAAUCUUGGUGAUGCUGACA 3′  69 IL4Rα #35(S): 5′ UAAGAAAGAAUGGUGG 3′  70 IL4Rα #35(AS): 5′ CCACCAUUCUUUCUUAAUCUU 3′  71 IL4Rα #36(S): 5′ AAGAAAGAAUGGUGGG 3′  72 IL4Rα #36(AS): 5′ CCCACCAUUCUUUCUUAAUCU 3′  73 IL4Rα #37(S): 5′ AGAAAGAAUGGUGGGA 3′  74 IL4Rα #37(AS): 5′ UCCCACCAUUCUUUCUUAAUC 3′  75 IL4Rα #38(S): 5′ GAUUCCCAACCCAGCC 3′  76 IL4Rα #38(AS): 5′ GGCUGGGUUGGGAAUCUGAUC 3′  77 IL4Rα #39(S): 5′ AGCAAGACAGUCCUCU 3′  78 IL4Rα #39(AS): 5′ AGAGGACUGUCUUGCUGAUCU 3′  79 IL4Rα #40(S): 5′ GCAAGACAGUCCUCUG 3′  80 IL4Rα #40(AS): 5′ CAGAGGACUGUCUUGCUGAUC 3′  81 IL4Rα #41(S): 5′ CAAGACAGUCCUCUGG 3′  82 IL4Rα #41(AS): 5′ CCAGAGGACUGUCUUGCUGAU 3′  83 IL4Rα #42(S): 5′ GUUGUUUGAGGCCCCG 3′  84 IL4Rα #42(AS): 5′ CGGGGCCUCAAACAACUCCAC 3′  85 IL4Rα #43(S): 5′ AACAGAGAGCCUGUUC 3′  86 IL4Rα #43(AS): 5′ GAACAGGCUCUCUGUUAGCCG 3′  87 IL4Rα #44(S): 5′ CUGGGAGCAGAUCCUC 3′  88 IL4Rα #44(AS): 5′ GAGGAUCUGCUCCCAGGUUUC 3′  89 IL4Rα #45(S): 5′ CUAUCAGGAGUUUGUA 3′  90 IL4Rα #45(AS): 5′ UACAAACUCCUGAUAGCCACU 3′  91 IL4Rα #46(S): 5′ GGCUGGUUACAAGGCC 3′  92 IL4Rα #46(AS): 5′ GGCCUUGUAACCAGCCUCUCC 3′  93 IL4Rα #47(S): 5′ GCUGGUUACAAGGCCU 3′  94 IL4Rα #47(AS): 5′ AGGCCUUGUAACCAGCCUCUC 3′  95 IL4Rα #48(S): 5′ CUGGUUACAAGGCCUU 3′  96 IL4Rα #48(AS): 5′ AAGGCCUUGUAACCAGCCUCU 3′  97 IL4Rα #49(S): 5′ UGGUUACAAGGCCUUC 3′  98 IL4Rα #49(AS): 5′ GAAGGCCUUGUAACCAGCCUC 3′  99 IL4Rα #50(S): 5′ GGUUACAAGGCCUUCU 3′ 100 IL4Rα #50(AS): 5′ AGAAGGCCUUGUAACCAGCCU 3′ 101 IL4Rα #51(S): 5′ GUUACAAGGCCUUCUC 3′ 102 IL4Rα #51(AS): 5′ GAGAAGGCCUUGUAACCAGCC 3′ 103 IL4Rα #52(S): 5′ UUACAAGGCCUUCUCA 3′ 104 IL4Rα #52(AS): 5′ UGAGAAGGCCUUGUAACCAGC 3′ 105 IL4Rα #53(S): 5′ GUGCGGCCACCUGAAA 3′ 106 IL4Rα #53(AS): 5′ UUUCAGGUGGCCGCACAGGUG 3′ 107 IL4Rα #54(S): 5′ GCUGUGGCUGCUGCUG 3′ 108 IL4Rα #54(AS): 5′ CAGCAGCAGCCACAGCAAGGA 3′ 109 IL4Rα #55(S): 5′ AGCCGAGCCUAGAAAC 3′ 110 IL4Rα #55(AS): 5′ GUUUCUAGGCUCGGCUUCUAG 3′ 111 IL4Rα #56(S): 5′ GGGAACAUGAAGGUCU 3′ 112 IL4Rα #56(AS): 5′ AGACCUUCAUGUUCCCAGAGC 3′ 113 IL4Rα #57(S): 5′ CUUGCAGGAGCCCACC 3′ 114 IL4Rα #57(AS): 5′ GGUGGGCUCCUGCAAGACCUU 3′ 115 IL4Rα #58(S): 5′ UUGCAGGAGCCCACCU 3′ 116 IL4Rα #58(AS): 5′ AGGUGGGCUCCUGCAAGACCU 3′ 117 IL4Rα #59(S): 5′ AGUUCACACCAAUGUC 3′ 118 IL4Rα #59(AS): 5′ GACAUUGGUGUGAACUGUCAG 3′ 119 IL4Rα #60(S): 5′ UUUCAGAAUCUAUAAC 3′ 120 IL4Rα #60(AS): 5′ GUUAUAGAUUCUGAAAUCUGC 3′ 121 IL4Rα #61(S): 5′ UAUAACGUGACCUACC 3′ 122 IL4Rα #61(AS): 5′ GGUAGGUCACGUUAUAGAUUC 3′ 123 IL4Rα #62(S): 5′ CACCUGGAGUGAGUGG 3′ 124 IL4Rα #62(AS): 5′ CCACUCACUCCAGGUGGUGUU 3′ 125 IL4Rα #63(S): 5′ ACCUGGAGUGAGUGGA 3′ 126 IL4Rα #63(AS): 5′ UCCACUCACUCCAGGUGGUGU 3′ 127 IL4Rα #64(S): 5′ UGUGCUAUGUCAGCAU 3′ 128 IL4Rα #64(AS): 5′ AUGCUGACAUAGCACAACAGG 3′ 129 IL4Rα #65(S): 5′ GUCAGCAUCACCAAGA 3′ 130 IL4Rα #65(AS): 5′ UCUUGGUGAUGCUGACAUAGC 3′ 131 IL4Rα #66(S): 5′ UCAGCAUCACCAAGAU 3′ 132 IL4Rα #66(AS): 5′ AUCUUGGUGAUGCUGACAUAG 3′ 133 IL4Rα #67(S): 5′ UGGUGGGAUCAGAUUC 3′ 134 IL4Rα #67(AS): 5′ GAAUCUGAUCCCACCAUUCUU 3′ 135 IL4Rα #68(S): 5′ GGUGGGAUCAGAUUCC 3′ 136 IL4Rα #68(AS): 5′ GGAAUCUGAUCCCACCAUUCU 3′ 137 IL4Rα #69(S): 5′ GUGCCCACACUGGAAG 3′ 138 IL4Rα #69(AS): 5′ CUUCCAGUGUGGGCACUUGGC 3′ 139 IL4Rα #70(S): 5′ CUGGAAGAAUUGUCUU 3′ 140 IL4Rα #70(AS): 5′ AAGACAAUUCUUCCAGUGUGG 3′ 141 IL4Rα #71(S): 5′ GUCCUCCAGCAUGGGG 3′ 142 IL4Rα #71(AS): 5′ CCCCAUGCUGGAGGACAUUUC 3′ 143 IL4Rα #72(S): 5′ AGUGGCUAUCAGGAGU 3′ 144 IL4Rα #72(AS): 5′ ACUCCUGAUAGCCACUGGUGG 3′ 145 IL4Rα #73(S): 5′ GUGGCUAUCAGGAGUU 3′ 146 IL4Rα #73(AS): 5′ AACUCCUGAUAGCCACUGGUG 3′

The asiRNAs listed in Table 1 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in annealing buffer (Bioneer Inc. Korea). Proper strand annealing was confirmed via gel electrophoresis using a UV transilluminator. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish. One day prior to transfection, 5×10³ A549 cells were seeded in 96-well plates. The A549 cells were transfected with 0.1 nM of the asiRNAs using RNAiMAX (Invitrogen Inc.) according to the manufacturer's instructions.

The IL4Rα mRNA levels in the transfected cells were measured 24 hours after transfection using qRT-PCR. Specifically, total RNA was extracted and synthesized into cDNA using Super Prep Cell Lysis & RT kit for qPCR (TOYOBO) according to manufacturer's instructions. Amplification of the IL4Rα gene was detected using IL4RαTaqMan® Probe (Hs00166237_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of IL4Rα inhibition by each of the 73 asiRNAs is depicted in FIG. 1. Fifteen of the asiRNA sequences, #5, #6, #20, #32, #38, #40, #41, #44, #48, #56, #58, #59, #64, #67 and #72, were selected for use in follow-up studies.

Example 2: Inhibition of IL4Rα mRNA Expression Using IL4Rα-Targeting asiRNAs

The asiRNA sequences selected in Example 1 were tested for their ability to inhibit IL4Rα mRNA expression.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in annealing buffer (Bioneer Inc. Korea). Proper strand annealing was confirmed via gel electrophoresis using a UV transilluminator. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with asiRNAs using RNAiMAX (Invitrogen Inc.) according to the manufacturer's instructions.

IL4Rα mRNA levels in A549 cells were determined using qRT-PCR 24 hours after asiRNA transfection. Specifically, total RNA was extracted using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the IL4Rα was detected using IL4Rα TaqMan® Probe (Hs00166237_m1). 18S RNA was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of IL4Rα inhibition of the 15 asiRNAs is provided in FIG. 2. AsiRNAs #5 and #6, which exhibited 40-50% inhibition of IL4Rα mRNA, were selected for use in follow-up studies.

Example 3: Inhibition of IL4Rα Protein Expression Using IL4Rα-Targeting asiRNAs

The two asiRNAs selected in Example 2 were tested for their ability to inhibit IL4Rα protein expression.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in annealing buffer (Bioneer Inc. Korea). Proper strand annealing was confirmed via gel electrophoresis using a UV transilluminator. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with 1 nM of the asiRNAs using RNAiMAX (Invitrogen Inc.) according to the manufacturer's instructions.

IL4Rα protein levels were determined via western blot 48 hours after asiRNA transfection. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 15 μg of the total protein extract was loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Bioworld) and then incubated overnight at 4° C. in 3% BSA containing anti-IL4Rα antibody (Acris) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The IL4Rα and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad). The results of the western blot are depicted in FIG. 3.

Example 4: Chemical Modification of asiRNAs for Self-Delivery

Chemical modifications were applied to the asiRNAs and the cellular delivery of the modified asiRNAs was tested in the absence of other delivery reagent. As described below, certain of the modifications improved endocytosis and stability of asiRNAs. Such cell penetrating asiRNAs (cp-asiRNAs) are able to be delivered into the cell in the absence of a delivery reagent.

Potential cp-asiRNA (Table 2) were screened for IL4Rα mRNA and protein inhibition in A549 cells. Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and IL4Rα expression levels were measured by qRT-PCR and western blot.

TABLE 2 Modified asiRNA sequences tested for self-delivery and IL4Rα inhibition. (m = 2′-O-Methyl RNA. *= phosphorothioate bond.) SEQ Name ID NO: SEQUENCE IL4Rα cp-asiRNA #5 (s) 147 5′ mUGmCGmUCmUCmCGmACmUA*mC*A*Cholesterol 3′ IL4Rα cp-asiRNA #5 21(2, 4) (AS) 148 5′ UGUAGUCGGAGACGmCmAG*G*U*G*G 3′ IL4Rα cp-asiRNA #6 (s) 149 5′ mGCmGUmCUmCCmGAmCUmAC*mA*U*Cholesterol 3′ IL4Rα cp-asiRNA #6 21(2, 4) (AS) 150 5′ AUGUAGUCGGAGACmGmCA*G*G*U*G 3′ IL4Rα cp-asiRNA #6 21(7, 4) (AS) 151 5′ AUGUAGUCGGAGACmGmCmA*mG*mG*mU*mG 3′

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish.

The potential cp-asiRNAs listed in Table 2 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in Opti-MEM (Gibco). Proper strand annealing of the potential cp-asiRNAs was confirmed by gel electrophoresis using a UV transilluminator.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (DMEM, Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at each point the cp-asiRNA containing OPTI-MEM media was replaced with a serum containing media.

Total RNA was extracted 48 hours after cp-asiRNA treatment, using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the IL4Rα was detected using IL4Rα TaqMan® Probe (Hs00166237_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

IL4Rα protein levels were determined via western blot 72 hours after cp-asiRNA treatment. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 15 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Biowolrd) and then incubated overnight at 4° C. in 3% BSA containing anti-IL4Rα antibody (Acris) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The IL4Rα and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

The levels of IL4Rα inhibition of three potential cp-asiRNAs is provided in FIG. 4 and FIG. 5. As the result, cp-asiRNA #5_21 (2, 4) and cp-asiRNA #6_21(2, 4) were selected for further studies.

Example 5: Additional Chemical Modification of Cp-asiRNA Structures

Other potential IL4Rα cp-asiRNA structures having different strand length was synthesized and tested for its ability to inhibit IL4Rα expression (Table 3).

TABLE 3 Additional cp-asiRNA sequence. SEQ Name ID NO: SEQUENCE IL4Rα cp-asiRNA #5 (s) 152 5′ mUGmCGmUCmUCmCGmACmUA*mC*A*Cholesterol 3′ IL4Rα cp-asiRNA #5 19(2, 4) (AS) 153 5′ UGUAGUCGGAGACGmC*mA*G*G*U 3′ IL4Rα cp-asiRNA #5 21(2, 4) (AS) 154 5′ UGUAGUCGGAGACGmCmAG*G*U*G*G 3′ IL4Rα cp-asiRNA #6 (s) 155 5′ mGCmGUmCUmCCmGAmCUmAC*mA*U*Cholesterol 3′ IL4Rα cp-asiRNA #6 19(2, 4) (AS) 156 5′ AUGUAGUCGGAGACmG*mC*A*G*G 3′ IL4Rα cp-asiRNA #6 21 (2, 4) (AS) 157 5′ AUGUAGUCGGAGACmGmCA*G*G*U*G 3′ (m = 2′-O-Methyl RNA. *= phosphorothioate bond.)

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish.

The potential cp-asiRNAs listed in Table 3 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in Opti-MEM (Gibco). Proper strand annealing of the potential cp-asiRNAs was confirmed by gel electrophoresis using a UV transilluminator.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (DMEM, Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at each point the cp-asiRNA containing OPTI-MEM media was replaced with a serum containing media.

Total RNA was extracted 48 hours after cp-asiRNA treatment, using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the IL4Rα was detected using IL4RαTaqMan® Probe (Hs00166237_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

IL4Rα protein levels were determined via western blot 72 hours after cp-asiRNA treatment. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 15 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Bioworld) and then incubated overnight at 4° C. in 3% BSA containing anti-IL4Rα antibody (Acris) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The IL4Rα and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

As shown the FIG. 6 and FIG. 7, cp-asiRNAs with different antisense strand lengths (21 or 19 nucleotides) exhibited the similar mRNA levels of IL4Rα inhibition.

Example 6: Screening for TRPA1-Specific Asymmetric Shorter-Duplex Small Interfering RNAs

To identify asymmetric shorter-duplex small interfering RNAs (asiRNAs) that inhibit TRPA1 with high efficiency, 102 asiRNAs were synthesized and screened. The nucleic acid sequences of the screened asiRNAs are provided in Table 4.

TABLE 4 Nucleic acid sequences for exemplary TRPA1-targeting asiRNA. SEQ ID NO: SEQUENCE 158 TRPA1#1(S): 5′ UGAAGGACGCUCUCCA 3′ 159 TRPA1#1(AS): 5′ UGGAGAGCGUCCUUCAGAAUC 3′ 160 TRPA1#2(S): 5′ GAAGGACGCUCUCCAC 3′ 161 TRPA1#2(AS): 5′ GUGGAGAGCGUCCUUCAGAAU 3′ 162 TRPA1#3(S): 5′ UGAAGGACGCUCUCCA 3′ 163 TRPA1#3(AS): 5′ UGGAGAGCGUCCUUCAGAAUC 3′ 164 TRPA1#4(S): 5′ AGGACGCUCUCCACUU 3′ 165 TRPA1#4(AS): 5′ AAGUGGAGAGCGUCCUUCAGA 3′ 166 TRPA1#5(S): 5′ GGACGCUCUCCACUUA 3′ 167 TRPA1#5(AS): 5′ UAAGUGGAGAGCGUCCUUCAG 3′ 168 TRPA1#6(S): 5′ GACGCUCUCCACUUAU 3′ 169 TRPA1#6(AS): 5′ AUAAGUGGAGAGCGUCCUUCA 3′ 170 TRPA1#7(S): 5′ UUUUGCAGCCAGUUAU 3′ 171 TRPA1#7(AS): 5′ AUAACUGGCUGCAAAAUGCAG 3′ 172 TRPA1#8(S): 5′ UUUGCAGCCAGUUAUG 3′ 173 TRPA1#8(AS): 5′ CAUAACUGGCUGCAAAAUGCA 3′ 174 TRPA1#9(S): 5′ UUGCAGCCAGUUAUGG 3′ 175 TRPA1#9(AS): 5′ CCAUAACUGGCUGCAAAAUGC 3′ 176 TRPA1#10(S): 5′ UGCAGCCAGUUAUGGG 3′ 177 TRPA1#10(AS): 5′ CCCAUAACUGGCUGCAAAAUG 3′ 178 TRPA1#11(S): 5′ GCAGCCAGUUAUGGGC 3′ 179 TRPA1#11(AS): 5′ GCCCAUAACUGGCUGCAAAAU 3′ 180 TRPA1#12(S): 5′ CAGCCAGUUAUGGGCG 3′ 181 TRPA1#12(AS): 5′ CGCCCAUAACUGGCUGCAAAA 3′ 182 TRPA1#13(S): 5′ CAUAAGUGAUACGAGG 3′ 183 TRPA1#13(AS): 5′ CCUCGUAUCACUUAUGUCUUG 3′ 184 TRPA1#14(S): 5′ AUAAGUGAUACGAGGC 3′ 185 TRPA1#14(AS): 5′ AAGACAUAAGUGAUACGAGGC 3′ 186 TRPA1#15(S): 5′ UAAGUGAUACGAGGCU 3′ 187 TRPA1#15(AS): 5′ AGCCUCGUAUCACUUAUGUCU 3′ 188 TRPA1#16(S): 5′ AAGUGAUACGAGGCUU 3′ 189 TRPA1#16(AS): 5′ AAGCCUCGUAUCACUUAUGUC 3′ 190 TRPA1#17(S): 5′ CAGUGACCACAAUGGC 3′ 191 TRPA1#17(AS): 5′ GCCAUUGUGGUCACUGAGAAA 3′ 192 TRPA1#18(S): 5′ AGUGACCACAAUGGCU 3′ 193 TRPA1#18(AS): 5′ AGCCAUUGUGGUCACUGAGAA 3′ 194 TRPA1#19(S): 5′ GUGACCACAAUGGCUG 3′ 195 TRPA1#19(AS): 5′ CAGCCAUUGUGGUCACUGAGA 3′ 196 TRPA1#20(S): 5′ UGACCACAAUGGCUGG 3′ 197 TRPA1#20(AS): 5′ CCAGCCAUUGUGGUCACUGAG 3′ 198 TRPA1#21(S): 5′ GACCACAAUGGCUGGA 3′ 199 TRPA1#21(AS): 5′ UCCAGCCAUUGUGGUCACUGA 3′ 200 TRPA1#22(S): 5′ ACCACAAUGGCUGGAC 3′ 201 TRPA1#22(AS): 5′ GUCCAGCCAUUGUGGUCACUG 3′ 202 TRPA1#23(S): 5′ CACUCAGACCAUGAAG 3′ 203 TRPA1#23(AS): 5′ CUUCAUGGUCUGAGUGUACCC 3′ 204 TRPA1#24(S): 5′ ACUCAGACCAUGAAGG 3′ 205 TRPA1#24(AS): 5′ CCUUCAUGGUCUGAGUGUACC 3′ 206 TRPA1#25(S): 5′ CUCAGACCAUGAAGGU 3′ 207 TRPA1#25(AS): 5′ ACCUUCAUGGUCUGAGUGUAC 3′ 208 TRPA1#26(S): 5′ UCAGACCAUGAAGGUC 3′ 209 TRPA1#26(AS): 5′ GACCUUCAUGGUCUGAGUGUA 3′ 210 TRPA1#27(S): 5′ CAGACCAUGAAGGUCA 3′ 211 TRPA1#27(AS): 5′ UGACCUUCAUGGUCUGAGUGU 3′ 212 TRPA1#28(S): 5′ AGACCAUGAAGGUCAU 3′ 213 TRPA1#28(AS): 5′ AUGACCUUCAUGGUCUGAGUG 3′ 214 TRPA1#29(S): 5′ GACCAUGAAGGUCAUU 3′ 215 TRPA1#29(AS): 5′ AAUGACCUUCAUGGUCUGAGU 3′ 216 TRPA1#30(S): 5′ ACCAUGAAGGUCAUUC 3′ 217 TRPA1#30(AS): 5′ GAAUGACCUUCAUGGUCUGAG 3′ 218 TRPA1#31(S): 5′ CCAUGAAGGUCAUUCU 3′ 219 TRPA1#31(AS): 5′ AGAAUGACCUUCAUGGUCUGA 3′ 220 TRPA1#32(S): 5′ CAUGAAGGUCAUUCUU 3′ 221 TRPA1#32(AS): 5′ AAGAAUGACCUUCAUGGUCUG 3′ 222 TRPA1#33(S): 5′ AUGAAGGUCAUUCUUG 3′ 223 TRPA1#33(AS): 5′ CAAGAAUGACCUUCAUGGUCU 3′ 224 TRPA1#34(S): 5′ UGAAGGUCAUUCUUGA 3′ 225 TRPA1#34(AS): 5′ UCAAGAAUGACCUUCAUGGUC 3′ 226 TRPA1#35(S): 5′ GAAGGUCAUUCUUGAU 3′ 227 TRPA1#35(AS): 5′ AUCAAGAAUGACCUUCAUGGU 3′ 228 TRPA1#36(S): 5′ AAGGUCAUUCUUGAUA 3′ 229 TRPA1#36(AS): 5′ UAUCAAGAAUGACCUUCAUGG 3′ 230 TRPA1#37(S): 5′ AGGUCAUUCUUGAUAC 3′ 231 TRPA1#37(AS): 5′ GUAUCAAGAAUGACCUUCAUG 3′ 232 TRPA1#38(S): 5′ GGUCAUUCUUGAUACU 3′ 233 TRPA1#38(AS): 5′ AGUAUCAAGAAUGACCUUCAU 3′ 234 TRPA1#39(S): 5′ GUCAUUCUUGAUACUA 3′ 235 TRPA1#39(AS): 5′ UAGUAUCAAGAAUGACCUUCA 3′ 236 TRPA1#40(S): 5′ UCAUUCUUGAUACUAA 3′ 237 TRPA1#40(AS): 5′ UUAGUAUCAAGAAUGACCUUC 3′ 238 TRPA1#41(S): 5′ CAGAAGACAAGUCCUG 3′ 239 TRPA1#41(AS): 5′ CAGGACUUGUCUUCUGUGGAA 3′ 240 TRPA1#42(S): 5′ UUUCCAACAGAAAAGG 3′ 241 TRPA1#42(AS): 5′ CCUUUUCUGUUGGAAAAUUUG 3′ 242 TRPA1#43(S): 5′ GGCAAUGUGGAGCAAU 3′ 243 TRPA1#43(AS): 5′ AUUGCUCCACAUUGCCACUGC 3′ 244 TRPA1#44(S): 5′ GCAGGUGGAACUUCAU 3′ 245 TRPA1#44(AS): 5′ AUGAAGUUCCACCUGCAUAGC 3′ 246 TRPA1#45(S): 5′ CAGGUGGAACUUCAUA 3′ 247 TRPA1#45(AS): 5′ UAUGAAGUUCCACCUGCAUAG 3′ 248 TRPA1#46(S): 5′ AGGUGGAACUUCAUAC 3′ 249 TRPA1#46(AS): 5′ GUAUGAAGUUCCACCUGCAUA 3′ 250 TRPA1#47(S): 5′ GGUGGAACUUCAUACC 3′ 251 TRPA1#47(AS): 5′ GGUAUGAAGUUCCACCUGCAU 3′ 252 TRPA1#48(S): 5′ GUGGAACUUCAUACCA 3′ 253 TRPA1#48(AS): 5′ UGGUAUGAAGUUCCACCUGCA 3′ 254 TRPA1#49(S): 5′ UGAUUAUGGAAAUACC 3′ 255 TRPA1#49(AS): 5′ GGUAUUUCCAUAAUCAUCCAU 3′ 256 TRPA1#50(S): 5′ AAUACCCCUCUGCAUU 3′ 257 TRPA1#50(AS): 5′ AAUGCAGAGGGGUAUUUCCAU 3′ 258 TRPA1#51(S): 5′ UACCCCUCUGCAUUGU 3′ 259 TRPA1#51(AS): 5′ ACAAUGCAGAGGGGUAUUUCC 3′ 260 TRPA1#52(S): 5′ ACCCCUCUGCAUUGUG 3′ 261 TRPA1#52(AS): 5′ CACAAUGCAGAGGGGUAUUUC 3′ 262 TRPA1#53(S): 5′ UUGUGCUGUAGAAAAA 3′ 263 TRPA1#53(AS): 5′ UUUUUCUACAGCACAAUGCAG 3′ 264 TRPA1#54(S): 5′ ACGCUCUCCACUUAUA 3′ 265 TRPA1#54(AS): 5′ UAUAAGUGGAGAGCGUCCUUC 3′ 266 TRPA1#55(S): 5′ CCACUUAUAUUAGCAA 3′ 267 TRPA1#55(AS): 5′ UUGCUAAUAUAAGUGGAGAGC 3′ 268 TRPA1#56(S): 5′ GUGCCCAAGUAGACAU 3′ 269 TRPA1#56(AS): 5′ AUGUCUACUUGGGCACCUUUA 3′ 270 TRPA1#57(S): 5′ UGCCCAAGUAGACAUA 3′ 271 TRPA1#57(AS): 5′ UAUGUCUACUUGGGCACCUUU 3′ 272 TRPA1#58(S): 5′ GCCCAAGUAGACAUAA 3′ 273 TRPA1#58(AS): 5′ UUAUGUCUACUUGGGCACCUU 3′ 274 TRPA1#59(S): 5′ CCCAAGUAGACAUAAA 3′ 275 TRPA1#59(AS): 5′ UUUAUGUCUACUUGGGCACCU 3′ 276 TRPA1#60(S): 5′ CAAGUAGACAUAAAAG 3′ 277 TRPA1#60(AS): 5′ CUUUUAUGUCUACUUGGGCAC 3′ 278 TRPA1#61(S): 5′ AAGUAGACAUAAAAGA 3′ 279 TRPA1#61(AS): 5′ UCUUUUAUGUCUACUUGGGCA 3′ 280 TRPA1#62(S): 5′ AGUAGACAUAAAAGAU 3′ 281 TRPA1#62(AS): 5′ AUCUUUUAUGUCUACUUGGGC 3′ 282 TRPA1#63(S): 5′ AUUUAUGCAGAUGCAA 3′ 283 TRPA1#63(AS): 5′ UUGCAUCUGCAUAAAUUCAGG 3′ 284 TRPA1#64(S): 5′ UAUGGGCGUAUCAAUA 3′ 285 TRPA1#64(AS): 5′ UAUUGAUACGCCCAUAACUGG 3′ 286 TRPA1#65(S): 5′ AUGGGCGUAUCAAUAC 3′ 287 TRPA1#65(AS): 5′ GUAUUGAUACGCCCAUAACUG 3′ 288 TRPA1#66(S): 5′ CGAGGCUUCUGAAUGA 3′ 289 TRPA1#66(AS): 5′ UCAUUCAGAAGCCUCGUAUCA 3′ 290 TRPA1#67(S): 5′ GAGGCUUCUGAAUGAA 3′ 291 TRPA1#67(AS): 5′ UUCAUUCAGAAGCCUCGUAUC 3′ 292 TRPA1#68(S): 5′ AGGCUUCUGAAUGAAG 3′ 293 TRPA1#68(AS): 5′ CUUCAUUCAGAAGCCUCGUAU 3′ 294 TRPA1#69(S): 5′ UCUCAGUGACCACAAU 3′ 295 TRPA1#69(AS): 5′ AUUGUGGUCACUGAGAAACAA 3′ 296 TRPA1#70(S): 5′ CUCAGUGACCACAAUG 3′ 297 TRPA1#70(AS): 5′ CAUUGUGGUCACUGAGAAACA 3′ 298 TRPA1#71(S): 5′ ACACUCAGACCAUGAA 3′ 299 TRPA1#71(AS): 5′ UUCAUGGUCUGAGUGUACCCG 3′ 300 TRPA1#72(S): 5′ ACUGUCUUGGUCUCAU 3′ 301 TRPA1#72(AS): 5′ AUGAGACCAAGACAGUAAGAU 3′ 302 TRPA1#73(S): 5′ CUGUCUUGGUCUCAUA 3′ 303 TRPA1#73(AS): 5′ UAUGAGACCAAGACAGUAAGA 3′ 304 TRPA1#74(S): 5′ UGUCUUGGUCUCAUAC 3′ 305 TRPA1#74(AS): 5′ GUAUGAGACCAAGACAGUAAG 3′ 306 TRPA1#75(S): 5′ AUAUUUGGGUAUUGCA 3′ 307 TRPA1#75(AS): 5′ UGCAAUACCCAAAUAUACUUG 3′ 308 TRPA1#76(S): 5′ GGGUAUUGCAAAGAAG 3′ 309 TRPA1#76(AS): 5′ CUUCUUUGCAAUACCCAAAUA 3′ 310 TRPA1#77(S): 5′ UUUUCCAACAGAAAAG 3′ 311 TRPA1#77(AS): 5′ CUUUUCUGUUGGAAAAUUUGC 3′ 312 TRPA1#78(S): 5′ GCAAUGUGGAGCAAUU 3′ 313 TRPA1#78(AS): 5′ AAUUGCUCCACAUUGCCACUG 3′ 314 TRPA1#79(S): 5′ UUUUGGACUCAGCUUU 3′ 315 TRPA1#79(AS): 5′ AAAGCUGAGUCCAAAAGCCAG 3′ 316 TRPA1#80(S): 5′ UUUGGACUCAGCUUUU 3′ 317 TRPA1#80(AS): 5′ AAAAGCUGAGUCCAAAAGCCA 3′ 318 TRPA1#81(S): 5′ UUGGACUCAGCUUUUA 3′ 319 TRPA1#81(AS): 5′ UAAAAGCUGAGUCCAAAAGCC 3′ 320 TRPA1#82(S): 5′ CUAGGAGAUAUCAAUU 3′ 321 TRPA1#82(AS): 5′ AAUUGAUAUCUCCUAGCAUCA 3′ 322 TRPA1#83(S): 5′ UAGGAGAUAUCAAUUA 3′ 323 TRPA1#83(AS): 5′ UAAUUGAUAUCUCCUAGCAUC 3′ 324 TRPA1#84(S): 5′ GGAGAUAUCAAUUAUC 3′ 325 TRPA1#84(AS): 5′ GAUAAUUGAUAUCUCCUAGCA 3′ 326 TRPA1#85(S): 5′ GAGAUAUCAAUUAUCG 3′ 327 TRPA1#85(AS): 5′ CGAUAAUUGAUAUCUCCUAGC 3′ 328 TRPA1#86(S): 5′ AGAUAUCAAUUAUCGA 3′ 329 TRPA1#86(AS): 5′ UCGAUAAUUGAUAUCUCCUAG 3′ 330 TRPA1#87(S): 5′ AUAUUUGUCCCAAUUG 3′ 331 TRPA1#87(AS): 5′ CAAUUGGGACAAAUAUUGUGA 3′ 332 TRPA1#88(S): 5′ UAUUUGUCCCAAUUGU 3′ 333 TRPA1#88(AS): 5′ ACAAUUGGGACAAAUAUUGUG 3′ 334 TRPA1#89(S): 5′ CCAAUUGUCCUCAUGA 3′ 335 TRPA1#89(AS): 5′ UCAUGAGGACAAUUGGGACAA 3′ 336 TRPA1#90(S): 5′ CAAUUGUCCUCAUGAA 3′ 337 TRPA1#90(AS): 5′ UUCAUGAGGACAAUUGGGACA 3′ 338 TRPA1#91(S): 5′ UGCUGAGGUCCAGAAA 3′ 339 TRPA1#91(AS): 5′ UUUCUGGACCUCAGCAAUGUC 3′ 340 TRPA1#92(S): 5′ AGAGGAUAGCUAUGCA 3′ 341 TRPA1#92(AS): 5′ UGCAUAGCUAUCCUCUUCAAU 3′ 342 TRPA1#93(S): 5′ GAGGAUAGCUAUGCAG 3′ 343 TRPA1#93(AS): 5′ CUGCAUAGCUAUCCUCUUCAA 3′ 344 TRPA1#94(S): 5′ UAUGCAGGUGGAACUU 3′ 345 TRPA1#94(AS): 5′ AAGUUCCACCUGCAUAGCUAU 3′ 346 TRPA1#95(S): 5′ AUGCAGGUGGAACUUC 3′ 347 TRPA1#95(AS): 5′ GAAGUUCCACCUGCAUAGCUA 3′ 348 TRPA1#96(S): 5′ UGCAGGUGGAACUUCA 3′ 349 TRPA1#96(AS): 5′ UGAAGUUCCACCUGCAUAGCU 3′ 350 TRPA1#97(S): 5′ AACAGCAUGAGCUCAU 3′ 351 TRPA1#97(AS): 5′ AUGAGCUCAUGCUGUUUUUCC 3′ 352 TRPA1#98(S): 5′ CAGAAGAUGGAGAUCA 3′ 353 TRPA1#98(AS): 5′ UGAUCUCCAUCUUCUGAAUGA 3′ 354 TRPA1#99(S): 5′ AGAAGAUGGAGAUCAU 3′ 355 TRPA1#99(AS): 5′ AUGAUCUCCAUCUUCUGAAUG 3′ 356 TRPA1#100(S): 5′ GAAGAUGGAGAUCAUC 3′ 357 TRPA1#100(AS): 5′ GAUGAUCUCCAUCUUCUGAAU 3′ 358 TRPA1#101(S): 5′ AAGAUGGAGAUCAUCU 3′ 359 TRPA1#101(AS): 5′ AGAUGAUCUCCAUCUUCUGAA 3′ 360 TRPA1#102(S): 5′ GAUGGAGAUCAUCUCU 3′ 361 TRPA1#102(AS): 5′ AGAGAUGAUCUCCAUCUUCUG 3′

The asiRNAs listed in Table 4 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in annealing buffer (Bioneer Inc. Korea). Proper strand annealing was confirmed via gel electrophoresis using a UV transilluminator. For the screen, 5×10³ A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish were seed 96 well plates. The A549 cells were transfected with 0.1 nM of the asiRNAs using RNAiMAX (Invitrogen Inc.) according to the manufacturer's instructions.

The TRPA1 mRNA levels in the transfected cells were measured 24 hours after transfection using qRT-PCR. Specifically, total RNA was extracted and synthesized the cDNA using SuperPrep Cell Lysis & RT kit for qPCR (TOYOBO) according to manufacturer's instructions. qRT-PCR was performed using THUNDERBIRD® Probe qPCR Mix (TOYOBO) according to manufacturer's instructions. Amplification of the TRPA1 was detected using TRPA1 TaqMan® Probe (Hs00175798_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of TRPA1 inhibition by each of the 102 asiRNAs is provided in FIG. 9. 14 of the asiRNA sequences, asiRNA (#32), asiRNA (#34), asiRNA (#35), asiRNA (#38), asiRNA (#40), asiRNA (#41), asiRNA (#50), asiRNA (#64), asiRNA (#66), asiRNA (#69), asiRNA (#71), asiRNA (#72), asiRNA (#78) and asiRNA (#81), were selected for use in follow-up studies.

Example 7: Inhibition of TRPA1 mRNA and Protein Expression Using TRPA1-Targeting asiRNAs

The asiRNAs selected in Example 6, asiRNA (#32), asiRNA (#34), asiRNA (#35), asiRNA (#38), asiRNA (#40), asiRNA (#41), asiRNA (#50), asiRNA (#64), asiRNA (#66), asiRNA (#69), asiRNA (#71), asiRNA (#72), asiRNA (#78) and asiRNA (#81), were tested for their ability to inhibit TRPA1 mRNA and protein expression.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in annealing buffer (Bioneer Inc. Korea). Proper strand annealing was confirmed via gel electrophoresis using a UV transilluminator. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with 1 nM of the asiRNAs using RNAiMAX (Invitrogen Inc.) according to the manufacturer's instructions.

24 hours after asiRNA transfection, total RNA was extracted using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the TRPA1 was detected using TRPA1 TaqMan® Probe (Hs00175798_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

48 hours after asiRNA transfection, TRPA1 protein levels were determined via western blot. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 30 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 5% skim milk (Seoul Milk) and 1% BSA (Bioworld) and then incubated overnight at 4° C. in 5% skim milk and 1% BSA containing anti-TRPA1 antibody (Novus) and anti-β-actin antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The TRPA1 and β-actin bands were then imaged using a Chemidoc instrument (Bio-rad).

The level of TRPA1 inhibition of 14 asiRNAs is provided in FIG. 10. The results of western blot are depicted in FIG. 11. asiRNA (#71) and asiRNA (#81) were selected for use in follow-up studies.

Example 8: Chemical Modification of asiRNAs for Self-Delivery

Chemical modifications were applied to the asiRNAs and the cellular delivery of the modified asiRNAs was tested in the absence of other delivery reagents. As described below, certain of the modifications improved endocytosis and stability of asiRNAs. Such cell penetrating asiRNAs (cp-asiRNAs) are able to be delivered into the cell in the absence of a delivery reagent.

Potential cp-asiRNA (Table 5) screened for TRPA1 mRNA and protein inhibition in A549 cells. Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and TRPA1 expression levels were measured by qRT-PCR and western blot.

TABLE 5 Modified asiRNA sequences tested for self-delivery and TRPA1 inhibition. SEQ Name ID NO: SEQUENCE TRPA1 cp-asiRNA 362 5′ mACmACmUCmAGmACmCAmU*G*mA*A*Cholesterol 3′ #71 PS4 (s) TRPA1 cp-asiRNA 363 5′ UUCAUGGUCUGAGUmGmUA*C*C*C*G 3′ #71 21(2, 4) (AS) TRPA1 cp-asiRNA 364 5′ UUCAUGGUCUGAGUmGmUmA*mC*C*C*G 3′ #71 21(4, 4) (AS) TRPA1 cp-asiRNA 365 5′ UUCAUGGUCUGAGUmGmUmA*mC*mC*mC*mG 3′ #71 21(7, 4) (AS) TRPA1 cp-asiRNA 366 5′ mUUmGGmACmUCmAGmCUmU*UmU*A*Cholesterol 3′ #81 PS4 (s) TRPA1 cp-asiRNA 367 5′ UAAAAGCUGAGUCCmAmAA*A*G*C*C 3′ #81 21(2, 4) (AS) TRPA1 cp-asiRNA 368 5′ UAAAAGCUGAGUCCmAmAmA*mA*G*C*C 3′ #81 21(4, 4) (AS) TRPA1 cp-asiRNA 369 5′ UAAAAGCUGAGUCCmAmAmA*mA*mG*mC*mC 3′ #81 21(7, 4) (AS) (m = 2′-O-Methyl RNA. *= phosphorothioate bond.)

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin and 100 μg/ml Streptomycin in a 100 mm cell culture dish.

The potential cp-asiRNAs listed in Table 2 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in Opti-MEM (Gibco). Proper strand annealing of the potential cp-asiRNAs was confirmed by gel electrophoresis using a UV transilluminator.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (DMEM, Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at each point the cp-asiRNA containing OPTI-MEM media was replaced with a serum containing media.

Total RNA was extracted 48 hours after asiRNA transfection, using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the TRPA1 was detected using TRPA1 TaqMan® Probe (Hs00175798_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

TRPA1 protein levels were determined via western blot 72 hours after asiRNA transfection. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH8.0). 30 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 5% skim milk (Seoul Milk) and 1% BSA (Bioworld) and then incubated overnight at 4° C. in 5% skim milk and 1% BSA containing anti-TRPA1 antibody (Novus) and anti-β-actin antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The TRPA1 and β-actin bands were then imaged using a Chemidoc instrument (Bio-rad).

The level of TRPA1 inhibition of 6 potential cp-asiRNAs is provided in FIG. 12 and FIG. 13. cp-asiRNA #71_21(4, 4) and cp-asiRNA #81_21 (4, 4) was selected for further studies.

Example 9: Additional Chemical Modification of Cp-asiRNA Structures

A variety of potential TRPA1 cp-asiRNA structures having different strand length and number of phosphorothioate bond and 2′-O-methylation modifications were synthesized and tested for its ability to inhibit TRPA1 expression (Table 6).

TABLE 6 Additional cp-asiRNA sequence. Name SEQ ID NO: SEQUENCE TRPA1 cp-asiRNA 370 5′ mACmACmUCmAGmACmCAmUG*mA*A*Cholesterol 3′ #71_PS3 (s) TRPA1 cp-asiRNA 371 5′ mACmACmUCmAGmACmCAmU*G*mA*A*Cholesterol 3′ #71_PS4 (s) TRPA1 cp-asiRNA 372 5′ UUCAUGGUCUGAGUmG*mU*mA*mC*C 3′ #71_19(4, 4) (AS) TRPA1 cp-asiRNA 373 5′ UUCAUGGUCUGAGUmGmUmA*mC*C*C*G 3′ #71_21(4, 4) (AS) TRPA1 cp-asiRNA 374 5′ mUUmGGmACmUCmAGmCUmUU*mU*A*Cholesterol 3′ #81_PS3 (s) TRPA1 cp-asiRNA 375 5′ mUUmGGmACmUCmAGmCUmU*U*mU*A*Cholesterol 3′ #81_PS4 (s) TRPA1 cp-asiRNA 376 5′ UAAAAGCUGAGUCCmA*mA*mA*mA*G 3′ #81_19(4, 4) (AS) TRPA1 cp-asiRNA 377 5′ UAAAAGCUGAGUCCmAmAmA*mA*G*C*C 3′ #81_21(4, 4) (AS) (m = 2′-O-Methyl RNA. *= phosphorothioate bond.)

The ability of 1 μM or 3 μM of each of the potential cp-asiRNAs listed in Table 6 to inhibit TRPA1 mRNA and protein expression in A549 cells was tested.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), 100 units/ml Penicillin and 100 μg/ml Streptomycin. The potential cp-asiRNAs listed in Table 3 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in Opti-MEM (Gibco). Proper strand annealing was confirmed by gel electrophoresis using a UV transilluminator.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with DMEM (Gibco) then cultured in the presence of the potential cp-asiRNAs in Opti-MEM media for 24 hours, at which point the cp-asiRNA-containing Opti-MEM media was replaced with a serum-containing media.

Total RNA was extracted 48 hours after asiRNA transfection using RNAiPlus® (TaKaRa) and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the TRPA1 was detected using TRPA1 TaqMan® Probe (Hs00175798_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

TRPA1 protein levels were determined via western blot 72 hours after asiRNA transfection. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 30 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 5% skim milk (Seoul Milk) and 1% BSA (Bioworld) and then incubated overnight at 4° C. in 5% skim milk and 1% BSA containing anti-TRPA1 antibody (Novus) and anti-β-actin antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The TRPA1 and β-actin bands were then imaged using a Chemidoc instrument (Bio-rad).

As shown the FIG. 14 and FIG. 15, the indicated TRPA1 cp-asiRNA exhibited the similar mRNA levels of TRPA1 inhibition.

Example 10: Inhibition of TRPA1 Protein Expression Using TRPA1-Specific Cp-asiRNAs

The efficacy of cp-asiRNAs for the inhibition of TRPA1 protein level was tested.

Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and TRPA1 protein levels were measured by western blot.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 100 units/ml Penicillin and 100 μg/ml Streptomycin. The potential cp-asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in Opti-MEM (Gibco). Proper strand annealing was confirmed by gel electrophoresis using a UV transilluminator.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with DMEM (Gibco) then cultured in the presence of the potential cp-asiRNAs in Opti-MEM media for 24 hours, at which point the cp-asiRNA-containing Opti-MEM media was replaced with a serum-containing media.

TRPA1 protein levels were determined via western blot 72 hours after asiRNA transfection. Briefly, the transfected A549 cells were lysed with 1% SDS lysis buffer (1% SDS, 100 mM Tris pH 8.0). 30 μg of the total protein extract of A549 cells were loaded onto an 8% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 5% skim milk (Seoul Milk) and 1% BSA (Bioworld) and then incubated overnight at 4° C. in 5% skim milk and 1% BSA containing anti-TRPA1 antibody (Novus) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 5% skim milk with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL substrate (Thermo scientific) for 1 minute. The TRPA1 and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

The results of the western blot assay are depicted in FIG. 16. As a result, TRPA1 cp-asiRNA #81 containing 3 phosphorothioate bond on sense strand and 19 nucleotides antisense strand with 4 phosphorothioate bond and four 2′-O-Methylation (TRPA1 cp-asiRNA #81_PS3/19(4,4)) exhibited the highest levels of TRPA1 inhibition.

Example 11: Screening for F2RL1-Targeting Asymmetric Shorter-Duplex Small Interfering RNAs

To identify asymmetric shorter-duplex small interfering RNAs (asiRNAs) that inhibit F2RL1 with high efficiency, 100 asiRNAs were synthesized and screened. The nucleic acid sequences of the screened asiRNAs are provided in Table 7.

TABLE 7 Nucleic acid sequences for exemplary F2RL1-targeting asiRNA. SEQ ID NO: SEQUENCE 378 F2RL1#1(S): 5′CCUCUCUGUCAUCUGG 3′ 379 F2RL1#1(AS): 5′CCAGAUGACAGAGAGGAGGUC 3′ 380 F2RL1#2(S): 5′CUCUCUGUCAUCUGGU 3′ 381 F2RL1#2(AS): 5′ACCAGAUGACAGAGAGGAGGU 3′ 382 F2RL1#3(S): 5′UCUCUGUCAUCUGGUU 3′ 383 F2RL1#3(AS): 5′AACCAGAUGACAGAGAGGAGG 3′ 384 F2RL1#4(S): 5′CUCUGUCAUCUGGUUC 3′ 385 F2RL1#4(AS): 5′GAACCAGAUGACAGAGAGGAG 3′ 386 F2RL1#5(S): 5′UCUGUCAUCUGGUUCC 3′ 387 F2RL1#5(AS): 5′GGAACCAGAUGACAGAGAGGA 3′ 388 F2RL1#6(S): 5′CUGUCAUCUGGUUCCC 3′ 389 F2RL1#6(AS): 5′GGGAACCAGAUGACAGAGAGG 3′ 390 F2RL1#7(S): 5′UGUCAUCUGGUUCCCC 3′ 391 F2RL1#7(AS): 5′GGGGAACCAGAUGACAGAGAG 3′ 392 F2RL1#8(S): 5′CACCAUCCCUUUGUAU 3′ 393 F2RL1#8(AS): 5′AUACAAAGGGAUGGUGACCAG 3′ 394 F2RL1#9(S): 5′ACCAUCCCUUUGUAUG 3′ 395 F2RL1#9(AS): 5′CAUACAAAGGGAUGGUGACCA 3′ 396 F2RL1#10(S): 5′CCAUCCCUUUGUAUGU 3′ 397 F2RL1#10(AS): 5′ACAUACAAAGGGAUGGUGACC 3′ 398 F2RL1#11(S): 5′CAUCCCUUUGUAUGUC 3′ 399 F2RL1#11(AS): 5′GACAUACAAAGGGAUGGUGAC 3′ 400 F2RL1#12(S): 5′ACAAAGGGAUGGUGAC 3′ 401 F2RL1#12(AS): 5′GUCACCAUCCCUUUGUAUGUC 3′ 402 F2RL1#13(S): 5′UUCAAUUACUUCCUCU 3′ 403 F2RL1#13(AS) 5′AGAGGAAGUAAUUGAACAUGU 3′ 404 F2RL1#14(S): 5′UCAAUUACUUCCUCUC 3′ 405 F2RL1#14(AS): 5′GAGAGGAAGUAAUUGAACAUG 3′ 406 F2RL1#15(S): 5′CUUUGUCUAUUACUUU 3′ 407 F2RL1#15(AS): 5′AAAGUAAUAGACAAAGGGGUC 3′ 408 F2RL1#16(S): 5′UUUGUCUAUUACUUUG 3′ 409 F2RL1#16(AS): 5′CAAAGUAAUAGACAAAGGGGU 3′ 410 F2RL1#17(S): 5′UUGUCUAUUACUUUGU 3′ 411 F2RL1#17(AS): 5′ACAAAGUAAUAGACAAAGGGG 3′ 412 F2RL1#18(S): 5′AUGGCCAAUCUGGCCU 3′ 413 F2RL1#18(AS): 5′AGGCCAGAUUGGCCAUGUAAA 3′ 414 F2RL1#19(S): 5′UUGGCUGACCUCCUCU 3′ 415 F2RL1#19(AS): 5′AGAGGAGGUCAGCCAAGGCCA 3′ 416 F2RL1#20(S): 5′GGCUGACCUCCUCUCU 3′ 417 F2RL1#20(AS): 5′AGAGAGGAGGUCAGCCAAGGC 3′ 418 F2RL1#21(S): 5′GCUGACCUCCUCUCUG 3′ 419 F2RL1#21(AS): 5′CAGAGAGGAGGUCAGCCAAGG 3′ 420 F2RL1#22(S): 5′CUGACCUCCUCUCUGU 3′ 421 F2RL1#22(AS): 5′ACAGAGAGGAGGUCAGCCAAG 3′ 422 F2RL1#23(S): 5′UGACCUCCUCUCUGUC 3′ 423 F2RL1#23(AS) 5′GACAGAGAGGAGGUCAGCCAA 3′ 424 F2RL1#24(S): 5′GACCUCCUCUCUGUCA 3′ 425 F2RL1#24(AS): 5′UGACAGAGAGGAGGUCAGCCA 3′ 426 F2RL1#25(S): 5′ACCUCCUCUCUGUCAU 3′ 427 F2RL1#25(AS): 5′AUGACAGAGAGGAGGUCAGCC 3′ 428 F2RL1#26(S): 5′CCUCCUCUCUGUCAUC 3′ 429 F2RL1#26(AS): 5′GAUGACAGAGAGGAGGUCAGC 3′ 430 F2RL1#27(S): 5′CUCCUCUCUGUCAUCU 3′ 431 F2RL1#27(AS): 5′AGAUGACAGAGAGGAGGUCAG 3′ 432 F2RL1#28(S): 5′UCCUCUCUGUCAUCUG 3′ 433 F2RL1#28(AS): 5′CAGAUGACAGAGAGGAGGUCA 3′ 434 F2RL1#29(S): 5′GUCAUCUGGUUCCCCU 3′ 435 F2RL1#29(AS): 5′AGGGGAACCAGAUGACAGAGA 3′ 436 F2RL1#30(S): 5′ACAUGGCAACAACUGG 3′ 437 F2RL1#30(AS): 5′CCAGUUGUUGCCAUGUAUGUG 3′ 438 F2RL1#31(S): 5′UAUUGGCUUUUUCUAU 3′ 439 F2RL1#31(AS): 5′AUAGAAAAAGCCAAUAAGCAC 3′ 440 F2RL1#32(S): 5′AUUGGCUUUUUCUAUG 3′ 441 F2RL1#32(AS): 5′CAUAGAAAAAGCCAAUAAGCA 3′ 442 F2RL1#33(S): 5′UUGGCUUUUUCUAUGG 3′ 443 F2RL1#33 (AS): 5′CCAUAGAAAAAGCCAAUAAGC 3′ 444 F2RL1#34(S): 5′UUCUAUGGCAACAUGU 3′ 445 F2RL1#34(AS): 5′ACAUGUUGCCAUAGAAAAAGC 3′ 446 F2RL1#35(S): 5′UCUAUGGCAACAUGUA 3′ 447 F2RL1#35(AS): 5′UACAUGUUGCCAUAGAAAAAG 3′ 448 F2RL1#36(S): 5′CUCUUCAUGACCUGCC 3′ 449 F2RL1#36(AS): 5′GGCAGGUCAUGAAGAGAAUGG 3′ 450 F2RL1#37(S): 5′UCUUCAUGACCUGCCU 3′ 451 F2RL1#37(AS): 5′AGGCAGGUCAUGAAGAGAAUG 3′ 452 F2RL1#38(S): 5′CUUCAUGACCUGCCUC 3′ 453 F2RL1#38(AS): 5′GAGGCAGGUCAUGAAGAGAAU 3′ 454 F2RL1#39(S): 5′UUCAUGACCUGCCUCA 3′ 455 F2RL1#39(AS): 5′UGAGGCAGGUCAUGAAGAGAA 3′ 456 F2RL1#40(S): 5′UCAUGACCUGCCUCAG 3′ 457 F2RL1#40(AS): 5′CUGAGGCAGGUCAUGAAGAGA 3′ 458 F2RL1#41(S): 5′CAUGACCUGCCUCAGU 3′ 459 F2RL1#41(AS): 5′ACUGAGGCAGGUCAUGAAGAG 3′ 460 F2RL1#42(S): 5′UGCCUCAGUGUGCAGA 3′ 461 F2RL1#42(AS): 5′UCUGCACACUGAGGCAGGUCA 3′ 462 F2RL1#43(S): 5′GCCUCAGUGUGCAGAG 3′ 463 F2RL1#43(AS): 5′CUCUGCACACUGAGGCAGGUC 3′ 464 F2RL1#44(S): 5′CUCAGUGUGCAGAGGU 3′ 465 F2RL1#44(AS): 5′ACCUCUGCACACUGAGGCAGG 3′ 466 F2RL1#45(S): 5′UCAGUGUGCAGAGGUA 3′ 467 F2RL1#45(AS): 5′UACCUCUGCACACUGAGGCAG 3′ 468 F2RL1#46(S): 5′CAUCGUGAACCCCAUG 3′ 469 F2RL1#46(AS): 5′CAUGGGGUUCACGAUGACCCA 3′ 470 F2RL1#47(S): 5′AUCGUGAACCCCAUGG 3′ 471 F2RL1#47(AS): 5′CCAUGGGGUUCACGAUGACCC 3′ 472 F2RL1#48(S): 5′UCGUGAACCCCAUGGG 3′ 473 F2RL1#48(AS): 5′CCCAUGGGGUUCACGAUGACC 3′ 474 F2RL1#49(S): 5′CAGGAAGAAGGCAAAC 3′ 475 F2RL1#49(AS): 5′GUUUGCCUUCUUCCUGGAGUG 3′ 476 F2RL1#50(S): 5′GAAGAAGGCAAACA 3′ 477 F2RL1#50(AS): 5′UGUUUGCCUUCUUCCUGGAGU 3′ 478 F2RL1#51(S): 5′GGAAGAAGGCAAACAU 3′ 479 F2RL1#51(AS): 5′AUGUUUGCCUUCUUCCUGGAG 3′ 480 F2RL1#52(S): 5′GUCACCAUCCCUUUGU 3′ 481 F2RL1#52(AS): 5′ACAAAGGGAUGGUGACCAGCA 3′ 482 F2RL1#53(S): 5′UCACCAUCCCUUUGUA 3′ 483 F2RL1#53(AS): 5′UACAAAGGGAUGGUGACCAGC 3′ 484 F2RL1#54(S): 5′AUCCCUUUGUAUGUCG 3′ 485 F2RL1#54(AS): 5′CGACAUACAAAGGGAUGGUGA 3′ 486 F2RL1#55(S): 5′UGUAUGUCGUGAAGCA 3′ 487 F2RL1#55(AS): 5′UGCUUCACGACAUACAAAGGG 3′ 488 F2RL1#56(S): 5′GUAUGUCGUGAAGCAG 3′ 489 F2RL1#56(AS): 5′CUGCUUCACGACAUACAAAGG 3′ 490 F2RL1#57(S): 5′UAUGUCGUGAAGCAGA 3′ 491 F2RL1#57(AS): 5′UCUGCUUCACGACAUACAAAG 3′ 492 F2RL1#58(S): 5′GUCGUGAAGCAGACCA 3′ 493 F2RL1#58(AS): 5′UGGUCUGCUUCACGACAUACA 3′ 494 F2RL1#59(S): 5′UCGUGAAGCAGACCAU 3′ 495 F2RL1#59(AS): 5′AUGGUCUGCUUCACGACAUAC 3′ 496 F2RL1#60(S): 5′C GUGAAGCAGACCAUC 3′ 497 F2RL1#60(AS): 5′GAUGGUCUGCUUCACGACAUA 3′ 498 F2RL1#61(S): 5′GUGAAGCAGACCAUCU 3′ 499 F2RL1#61(AS): 5′AGAUGGUCUGCUUCACGACAU 3′ 500 F2RL1#62(S): 5′GGGAGACAUGUUCAAU 3′ 501 F2RL1#62(AS): 5′AUUGAACAUGUCUCCCACCAA 3′ 502 F2RL1#63(S): 5′GGAGACAUGUUCAAUU 3′ 503 F2RL1#63(AS): 5′AAUUGAACAUGUCUCCCACCA 3′ 504 F2RL1#64(S): 5′GAGACAUGUUCAAUUA 3′ 505 F2RL1#64(AS): 5′UAAUUGAACAUGUCUCCCACC 3′ 506 F2RL1#65(S): 5′AGACAUGUUCAAUUAC 3′ 507 F2RL1#65(AS): 5′GUAAUUGAACAUGUCUCCCAC 3′ 508 F2RL1#66(S): 5′GACAUGUUCAAUUACU 3′ 509 F2RL1#66(AS): 5′AGUAAUUGAACAUGUCUCCCA 3′ 510 F2RL1#67(S): 5′ACAUGUUCAAUUACUU 3′ 511 F2RL1#67(AS): 5′AAGUAAUUGAACAUGUCUCCC 3′ 512 F2RL1#68(S): 5′CAUGUUCAAUUACUUC 3′ 513 F2RL1#68(AS): 5′GAAGUAAUUGAACAUGUCUCC 3′ 514 F2RL1#69(S): 5′AUGUUCAAUUACUUCC 3′ 515 F2RL1#69(AS): 5′GGAAGUAAUUGAACAUGUCUC 3′ 516 F2RL1#70(S): 5′UGUUCAAUUACUUCCU 3′ 517 F2RL1#70(AS): 5′AGGAAGUAAUUGAACAUGUCU 3′ 518 F2RL1#71(S): 5′CAAUUACUUCCUCUCU 3′ 519 F2RL1#71(AS): 5′AGAGAGGAAGUAAUUGAACAU 3′ 520 F2RL1#72(S): 5′UUCCUCUCUCUGGCCA 3′ 521 F2RL1#72(AS): 5′UGGCCAGAGAGAGGAAGUAAU 3′ 522 F2RL1#73(S): 5′CCUCUCUCUGGCCAUU 3′ 523 F2RL1#73(AS): 5′AAUGGCCAGAGAGAGGAAGUA 3′ 524 F2RL1#74(S): 5′CUCUCUCUGGCCAUUG 3′ 525 F2RL1#74(AS): 5′CAAUGGCCAGAGAGAGGAAGU 3′ 526 F2RL1#75(S): 5′UCUCUCUGGCCAUUGG 3′ 527 F2RL1#75(AS): 5′CCAAUGGCCAGAGAGAGGAAG 3′ 528 F2RL1#76(S): 5′UGAAAACUCAGAGAAG 3′ 529 F2RL1#76(AS): 5′CUUCUCUGAGUUUUCAUCCAU 3′ 530 F2RL1#77(S): 5′GAAAACUCAGAGAAGA 3′ 531 F2RL1#77(AS): 5′UCUUCUCUGAGUUUUCAUCCA 3′ 532 F2RL1#78(S): 5′AAAACUCAGAGAAGAA 3′ 533 F2RL1#78(AS): 5′UUCUUCUCUGAGUUUUCAUCC 3′ 534 F2RL1#79(S): 5′AAACUCAGAGAAGAAA 3′ 535 F2RL1#79(AS): 5′UUUCUUCUCUGAGUUUUCAUC 3′ 536 F2RL1#80(S): 5′ACUCAGAGAAGAAAAG 3′ 537 F2RL1#80(AS): 5′CUUUUCUUCUCUGAGUUUUCA 3′ 538 F2RL1#81(S): 5′CUCAGAGAAGAAAAGG 3′ 539 F2RL1#81(AS): 5′CCUUUUCUUCUCUGAGUUUUC 3′ 540 F2RL1#82(S): 5′CUGCAUCGACCCCUUU 3′ 541 F2RL1#82(AS): 5′AAAGGGGUCGAUGCAGCUGUU 3′ 542 F2RL1#83(S): 5′UGCAUCGACCCCUUUG 3′ 543 F2RL1#83(AS): 5′CAAAGGGGUCGAUGCAGCUGU 3′ 544 F2RL1#84(S): 5′GCAUCGACCCCUUUGU 3′ 545 F2RL1#84(AS): 5′ACAAAGGGGUCGAUGCAGCUG 3′ 546 F2RL1#85(S): 5′CAUCGACCCCUUUGUC 3′ 547 F2RL1#85(AS): 5′GACAAAGGGGUCGAUGCAGCU 3′ 548 F2RL1#86(S): 5′AUCGACCCCUUUGUCU 3′ 549 F2RL1#86(AS): 5′AGACAAAGGGGUCGAUGCAGC 3′ 550 F2RL1#87(S): 5′UCGACCCCUUUGUCUA 3′ 551 F2RL1#87(AS): 5′UAGACAAAGGGGUCGAUGCAG 3′ 552 F2RL1#88(S): 5′CGACCCCUUUGUCUAU 3′ 553 F2RL1#88(AS): 5′AUAGACAAAGGGGUCGAUGCA 3′ 554 F2RL1#89(S): 5′GACCCCUUUGUCUAUU 3′ 555 F2RL1#89(AS): 5′AAUAGACAAAGGGGUCGAUGC 3′ 556 F2RL1#90(S): 5′ACCCCUUUGUCUAUUA 3′ 557 F2RL1#90(AS): 5′UAAUAGACAAAGGGGUCGAUG 3′ 558 F2RL1#91(S): 5′CCCCUUUGUCUAUUAC 3′ 559 F2RL1#91(AS): 5′GUAAUAGACAAAGGGGUCGAU 3′ 560 F2RL1#92(S): 5′CCCUUUGUCUAUUACU 3′ 561 F2RL1#92(AS): 5′AGUAAUAGACAAAGGGGUCGA 3′ 562 F2RL1#93(S): 5′CCUUUGUCUAUUACUU 3′ 563 F2RL1#93(AS): 5′AAGUAAUAGACAAAGGGGUCG 3′ 564 F2RL1#94(S): 5′UGUCUAUUACUUUGUU 3′ 565 F2RL1#94(AS): 5′AACAAAGUAAUAGACAAAGGG 3′ 566 F2RL1#95(S): 5′UGCCGAAGUGUCCGCA 3′ 567 F2RL1#95(AS): 5′UGCGGACACUUCGGCAAAGGA 3′ 568 F2RL1#96(S): 5′GCCGAAGUGUCCGCAC 3′ 569 F2RL1#96(AS): 5′GUGCGGACACUUCGGCAAAGG 3′ 570 F2RL1#97(S): 5′CCGAAGUGUCCGCACU 3′ 571 F2RL1#97(AS): 5′AGUGCGGACACUUCGGCAAAG 3′ 572 F2RL1#98(S): 5′CGAAGUGUCCGCACUG 3′ 573 F2RL1#98(AS): 5′CAGUGCGGACACUUCGGCAAA 3′ 574 F2RL1#99(S): 5′GAAGUGUCCGCACUGU 3′ 575 F2RL1#99(AS): 5′ACAGUGCGGACACUUCGGCAA 3′ 576 F2RL1#100(S): 5′AAGUGUCCGCACUGUA 3′ 577 F2RL1#100(AS): 5′UACAGUGCGGACACUUCGGCA 3′

The asiRNAs listed in Table 7 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in 1×siRNA duplex buffer (Bioneer). Proper strand annealing was confirmed via gel electrophoresis.

For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish. One day prior to transfection, 5×10³ A549 cells were seeded in 96-well plates. The A549 cells were transfected with 0.1 nM of the asiRNAs using RNAiMAX (Invitrogen) according to the manufacturer's instructions.

The F2RL1 mRNA levels in the transfected cells were measured 24 hours after transfection using real-time PCR. Specifically, total RNA was extracted and synthesized the cDNA using SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO), according to the manufacturer's instructions. Real-time PCR was performed using THUNDERBIRD® Probe qPCR Mix (TOYOBO) according to manufacturer's instructions. Amplification of the F2RL1 was detected using F2RL1 TaqMan® Probe (Hs00608346 m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of F2RL1 inhibition by each of the 100 asiRNAs is provided in FIG. 18. 29 of the asiRNA sequences, asiF2RL1#1, #22, #25, #26, #28, #29, #31, #34, #35, #45, #50, #51, #55, #57, #59, #64, #65, #67, #69, #73, #76, #77, #81, #84, #86, #87, #88, #92, and #100 were selected for use in follow-up studies.

Example 12: Inhibition of F2RL1 mRNA Expression Using F2RL1-Targeting asiRNAs

The 29 asiRNAs selected in Example 12, asiF2RL1 #1, #22, #25, #26, #28, #29, #31, #34, #35, #45, #50, #51, #55, #57, #59, #64, #65, #67, #69, #73, #76, #77, #81, #84, #86, #87, #88, #92, and #100, were tested for their ability to inhibit F2RL1 expression.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in 1×siRNA duplex buffer (Bioneer). Proper strand annealing was confirmed via gel electrophoresis. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with asiRNAs using RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Specifically, total RNA was extracted using RNAiso Plus (TaKaRa), and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the F2RL1 was detected using F2RL1 TaqMan® Probe (Hs00608346_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of F2RL1 inhibition of 29 asiRNAs is provided in FIG. 19. Twelve asiRNAs; asiF2RL1 #1, #22, #29, #50, #64, #67, #76, #77, #87, #88, #92, and #100 were selected for use in follow-up studies.

Example 13: Chemical Modification of asiRNAs

Chemical modifications were applied to 32 asiRNAs. As described below, certain of the modifications improved endocytosis and stability of the asiRNAs.

32 asiRNAs (Table 2) were tested for F2RL1 mRNA inhibition in A549 cells.

TABLE 8 Modified asiRNA sequences. SEQ ID NO: 578 F2RL1#29-1: (S) 5′ mGUmCAmUCmUGmGUmUCmCCmCU 3′ 579 F2RL1#29-1: (AS) 5′ AGGGGAACCAGAUGACAGAGA 3′ 580 F2RL1#29-2: (S) 5′ mGUmCAmUCmUGmGUmUCmCCmCU 3′ 581 F2RL1#29-2: (AS) 5′ AGGGGAACCAGAUGmAmCAGAGA 3′ 582 F2RL1#29-3: (S) 5′ mGUmCAmUCmUGmGUmUCmCCmCU 3′ 583 F2RL1#29-3: (AS) 5′ AGGGGAACCAGAUGmAmCmAmGAGA 3′ 584 F2RL1#29-4: (S) 5′ mGUmCAmUCmUGmGUmUCmCCmCU 3′ 585 F2RL1#29-4: (AS) 5′ AGGGGAACCAGAUGmAmCmAmGmAmGmA 3′ 586 F2RL1#50-1: (S) 5′ mAGmGAmAGmAAmGGmCAmAAmCA 3′ 587 F2RL1#50-1: (AS) 5′ UGUUUGCCUUCUUCCUGGAGU 3′ 588 F2RL1#50-2: (S) 5′ mAGmGAmAGmAAmGGmCAmAAmCA 3′ 589 F2RL1#50-2: (AS) 5′ UGUUUGCCUUCUUCmCmUGGAGU 3′ 590 F2RL1#50-3: (S) 5′ mAGmGAmAGmAAmGGmCAmAAmCA 3′ 591 F2RL1#50-3: (AS) 5′ UGUUUGCCUUCUUCmCmUmGmGAGU 3′ 592 F2RL1#50-4: (S) 5′ mAGmGAmAGmAAmGGmCAmAAmCA 3′ 593 F2RL1#50-4: (AS) 5′ UGUUUGCCUUCUUCmCmUmGmGmAmGmU 3′ 594 F2RL1#57-1: (S) 5′ mUAmUGmUCmGUmGAmAGmCAmGA 3′ 595 F2RL1#57-1: (AS) 5′ UCUGCUUCACGACAUACAAAG 3′ 596 F2RL1#57-2: (S) 5′ mUAmUGmUCmGUmGAmAGmCAmGA 3′ 597 F2RL1#57-2: (AS) 5′ UCUGCUUCACGACAmUmACAAAG 3′ 598 F2RL1#57-3: (S) 5′ mUAmUGmUCmGUmGAmAGmCAmGA 3′ 599 F2RL1#57-3: (AS) 5′ UCUGCUUCACGACAmUmAmCmAAAG 3′ 600 F2RL1#57-4: (S) 5′ mUAmUGmUCmGUmGAmAGmCAmGA 3′ 601 F2RL1#57-4: (AS) 5′ UCUGCUUCACGACAmUmAmCmAmAmAmG 3′ 602 F2RL1#64-1: (S) 5′ mGAmGAmCAmUGmUUmCAmAUmUA 3′ 603 F2RL1#64-1: (AS) 5′ UAAUUGAACAUGUCUCCCACC 3′ 604 F2RL1#64-2: (S) 5′ mGAmGAmCAmUGmUUmCAmAUmUA 3′ 605 F2RL1#64-2: (AS) 5′ UAAUUGAACAUGUCmUmCCCACC 3′ 606 F2RL1#64-3: (S) 5′ mGAmGAmCAmUGmUUmCAmAUmUA 3′ 607 F2RL1#64-3: (AS) 5′ UAAUUGAACAUGUCmUmCmCmCACC 3′ 608 F2RL1#64-4: (S) 5′ mGAmGAmCAmUGmUUmCAmAUmUA 3′ 609 F2RL1#64-4: (AS) 5′ UAAUUGAACAUGUCmUmCmCmCmAmCmC 3′ 610 F2RL1#67-1: (S) 5′ mACmAUmGUmUCmAAmUUmACmUU 3′ 611 F2RL1#67-1: (AS) 5′ AAGUAAUUGAACAUGUCUCCC 3′ 612 F2RL1#67-2: (S) 5′ mACmAUmGUmUCmAAmUUmACmUU 3′ 613 F2RL1#67-2: (AS) 5′ AAGUAAUUGAACAUmGmUCUCCC 3′ 614 F2RL1#67-3: (S) 5′ mACmAUmGUmUCmAAmUUmACmUU 3′ 615 F2RL1#67-3: (AS) 5′ AAGUAAUUGAACAUmGmUmCmUCCC 3′ 616 F2RL1#67-4: (S) 5′ mACmAUmGUmUCmAAmUUmACmUU 3′ 617 F2RL1#67-4: (AS) 5′ AAGUAAUUGAACAUmGmUmCmUmCmCmC 3′ 618 F2RL1#76-1: (S) 5′ mUGmAAmAAmCUmCAmGAmGAmAG 3′ 619 F2RL1#76-1: (AS) 5′ CUUCUCUGAGUUUUCAUCCAU 3′ 620 F2RL1#76-2: (S) 5′ mUGmAAmAAmCUmCAmGAmGAmAG 3′ 621 F2RL1#76-2: (AS) 5′ CUUCUCUGAGUUUUmCmAUCCAU 3′ 622 F2RL1#76-3: (S) 5′ mUGmAAmAAmCUmCAmGAmGAmAG 3′ 623 F2RL1#76-3: (AS) 5′ CUUCUCUGAGUUUUmCmAmUmCCAU 3′ 624 F2RL1#76-4: (S) 5′ mUGmAAmAAmCUmCAmGAmGAmAG 3′ 625 F2RL1#76-4: (AS) 5′ CUUCUCUGAGUUUUmCmAmUmCmCmAmU 3′ 626 F2RL1#77-1: (S) 5′ mGAmAAmACmUCmAGmAGmAAmGA 3′ 627 F2RL1#77-1: (AS) 5′ UCUUCUCUGAGUUUUCAUCCA 3′ 628 F2RL1#77-2: (S) 5′ mGAmAAmACmUCmAGmAGmAAmGA 3′ 629 F2RL1#77-2: (AS) 5′ UCUUCUCUGAGUUUmUmCAUCCA 3′ 630 F2RL1#77-3: (S) 5′ mGAmAAmACmUCmAGmAGmAAmGA 3′ 631 F2RL1#77-3: (AS) 5′ UCUUCUCUGAGUUUmUmCmAmUCCA 3′ 632 F2RL1#77-4: (S) 5′ mGAmAAmACmUCmAGmAGmAAmGA 3′ 633 F2RL1#77-4: (AS) 5′ UCUUCUCUGAGUUUmUmCmAmUmCmCmA 3′ 634 F2RL1#100-1: (S) 5′ mAAmGUmGUmCCmGCmACmUGmUA 3′ 635 F2RL1#100-1: (AS) 5′ UACAGUGCGGACACUUCGGCA 3′ 636 F2RL1#100-2: (S) 5′ mAAmGUmGUmCCmGCmACmUGmUA 3′ 637 F2RL1#100-2: (AS) 5′ UACAGUGCGGACACmUmUCGGCA 3′ 638 F2RL1#100-3: (S) 5′ mAAmGUmGUmCCmGCmACmUGmUA 3′ 639 F2RL1#100-3: (AS) 5′ UACAGUGCGGACACmUmUmCmGGCA 3′ 640 F2RL1#100-4: (S) 5′ mAAmGUmGUmCCmGCmACmUGmUA 3′ 641 F2RL1#100-4: (AS) 5′ UACAGUGCGGACACmUmUmCmGmGmCmA 3′ m = 2′-O-Methyl RNA

The asiRNAs listed in Table 8 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in 1×siRNA duplex buffer (Bioneer). Proper strand annealing was confirmed via gel electrophoresis. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with 0.3 nM of the asiRNAs using RNAiMAX (Invitrogen) according to the manufacturer's instructions.

The F2RL1 mRNA levels in the transfected cells were measured 24 hours after transfection using real-time PCR. Specifically, total RNA was extracted using RNAiso Plus(TaKaRa), and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. The synthesized cDNA was diluted and then real-time PCR was performed using THUNDERBIRD® Probe qPCR Mix (TOYOBO) according to manufacturer's instructions. Amplification of the F2RL1 was detected using F2RL1 TaqMan® Probe (Hs00608346_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of F2RL1 inhibition of 32 asiRNAs is provided in FIG. 20.

Example 14: Inhibition of F2RL1 mRNA Expression Using F2RL1-Targeting asiRNAs

The 12 asiRNAs selected in Example 12, asiF2RL1#1, #22, #29, #50, #64, #67, #76, #77, #87, #88, #92, and #100, were tested for their ability to inhibit F2RL1 expression.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in 1×siRNA duplex buffer (Bioneer). Proper strand annealing was confirmed via gel electrophoresis. For the screen, A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. The A549 cells were transfected with 1 nM of the asiRNAs using RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Specifically, total RNA was extracted using RNAiso Plus (TaKaRa), and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the F2RL1 was detected using F2RL1 TaqMan® Probe (Hs00608346_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of F2RL1 inhibition of 12 asiRNAs is provided in FIG. 21.

Example 15: Inhibition of F2RL1 Protein Expression Using F2RL1-Targeting asiRNAs

The efficacy of asiF2RL1 for the inhibition of F2RL1 protein was tested.

The asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in 1×siRNA duplex buffer (Bioneer). Proper strand annealing was confirmed via gel electrophoresis.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish. One day prior to transfection, 2.5×10⁴ A549 cells were seeded in 24-well plates. A549 cells were transfected with 1 nM of the asiRNAs using RNAiMAX (Invitrogen) according to the manufacturer's instructions.

The levels of F2RL1 protein expression were determined via western blot 72 hours after asiRNA transfection. Briefly, the transfected A549 cells were lysed with TX-100 lysis buffer (1% TX-100, 150 mM NaCl, 100 mM Tris (pH 8.8)). 10 μg of the total protein extracts of A549 cells were loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Bioworld) and then incubated overnight at 4° C. in 3% BSA containing anti-F2RL1 antibody (Abcam) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 1×TBST with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL for 1 minute. The F2RL1 and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

The results of the western blot assay are depicted in FIG. 22. asiF2RL1#22, #50, #77, and #92 were selected for the chemical modification.

Example 16: Chemical Modification of asiRNAs for Self-Delivery

Chemical modifications were applied to the 12 asiRNAs selected in Example 15 and the cellular delivery of the modified asiRNAs was tested in the absence of other delivery reagent. As described below, certain of the modifications improved endocytosis and stability of the asiRNAs. Such cell-penetrating asiRNAs (cp-asiRNAs) are able to be delivered into the cell in the absence of a delivery reagent.

12 potential cp-asiRNAs (Table 9) were screened for F2RL1 mRNA inhibition in A549 cells. Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and F2RL1 mRNA levels were measured by real-time PCR.

TABLE 9 Modified asiRNA sequences tested for self-delivery and F2RL1 inhibition. SEQ ID NO: 642 F2RL1#22-PS4/21(2, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ 643 F2RL1#22-PS4/21(2, 4) (AS): 5′ ACAGAGAGGAGGUCmAmGC*C*A*A*G 3′ 644 F2RL1#22-PS4/21(4, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ 645 F2RL1#22-P54/21(4, 4) (AS): 5′ ACAGAGAGGAGGUCmAmGmC*mC*A*A*G 3′ 646 F2RL1#22-PS4/21(7, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ 647 F2RL1#22-P54/21(7, 4) (AS): 5′ ACAGAGAGGAGGUCmAmGmC*mC*mA*mA*mG 3′ 648 F2RL1#50-PS4/21(2, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmA*A*mC*A*cholesterol 3′ 649 F2RL1#50-PS4/21(2, 4) (AS): 5′ UGUUUGCCUUCUUCmCmUG*G*A*G*U 3′ 650 F2RL1#50-PS4/21(4, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmA*A*mC*A*cholesterol 3′ 651 F2RL1#50-PS4/21(4, 4) (AS): 5′ UGUUUGCCUUCUUCmCmUmG*mG*A*G*U 3′ 652 F2RL1#50-PS4/21(7, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmA*A*mC*A*cholesterol 3′ 653 F2RL1#50-PS4/21(7, 4) (AS): 5′ UGUUUGCCUUCUUCmCmUmG*mG*mA*mG*mU 3′ 654 F2RL1#77-PS4/21(2, 4) (S): 5′ mGAmAAmACmUCmAGmAGmA*A*mG*A*cholesterol 3′ 655 F2RL1#77-PS4/21(2, 4) (AS): 5′ UCUUCUCUGAGUUUmUmCA*U*C*C*A 3′ 656 F2RL1#77-PS4/21(4, 4) (S): 5′ mGAmAAmACmUCmAGmAGmA*A*mG*A*cholesterol 3′ 657 F2RL1#77-PS4/21(4, 4) (AS): 5′ UCUUCUCUGAGUUUmUmCmA*mU*C*C*A 3′ 658 F2RL1#77-PS4/21(7, 4) (S): 5′ mGAmAAmACmUCmAGmAGmA*A*mG*A*cholesterol 3′ 659 F2RL1#77-PS4/21(7, 4) (AS): 5′ UCUUCUCUGAGUUUmUmCmA*mU*mC*mC*mA 3′ 660 F2RL1#92-PS4/21(2, 4) (S): 5′ mCCmCUmUUmGUmCUmAUmU*A*mC*U*cholesterol 3′ 661 F2RL1#92-P54/21(2, 4) (AS): 5′ AGUAAUAGACAAAGmGmGG*U*C*G*A 3′ 662 F2RL1#92-PS4/21(4, 4) (S): 5′ mCCmCUmUUmGUmCUmAUmU*A*mC*U*cholesterol 3′ 663 F2RL1#92-PS4/21(4, 4) (AS): 5′ AGUAAUAGACAAAGmGmGmG*mU*C*G*A 3′ 664 F2RL1#92-PS4/21(7, 4) (S): 5′ mCCmCUmUUmGUmCUmAUmU*A*mC*U*cholesterol 3′ 665 F2RL1#92-PS4/21(7, 4) (AS): 5′ AGUAAUAGACAAAGmGmGmG*mU*mC*mG*mA 3′ m = 2′-O-Methyl RNA, *= phosphorothioate bond.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish.

The potential cp-asiRNAs listed in Table 9 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing was confirmed via gel electrophoresis.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 8 and 24 hours, at each point the asiRNA-containing OPTI-MEM media was replaced with a serum-containing media.

The level of F2RL1 mRNA expression was determined using real-time PCR 48 hours after asiRNAs treatment. The level of F2RL1 inhibition of cp-asiRNAs is provided in FIG. 23.

Example 17: Inhibition of F2RL1 mRNA Expression Using F2RL1-Targeting Cp-asiRNAs

The efficacies of cp-asiRNAs for the inhibition of F2RL1 RNA were tested.

Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and F2RL1 mRNA levels were measured using real-time PCR.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish.

The cp-asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing was confirmed via gel electrophoresis.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at which point the asiRNA-containing OPTI-MEM media was replaced with a serum-containing media.

The level of F2RL1 mRNA expression was determined by real-time PCR 48 hours after asiRNA treatment. Total RNA was extracted using RNAiso Plus (TaKaRa), and then 500 ng of the extracted RNA was used for cDNA synthesis using the High-capacity cDNA reverse transcription kit (Applied Biosystems), according to the manufacturer's instructions. Amplification of the F2RL1 was detected using F2RL1 TaqMan® Probe (Hs00608346_m1). 18S was amplified as an internal control using 18S TaqMan® Probe (Hs03928985_g1).

The level of F2RL1 inhibition by cp-asiRNAs is provided in FIG. 24.

Example 18: Inhibition of F2RL1 Protein Using F2RL1-Targeting Cp-asiRNAs

The efficacies of cp-asiRNAs for the inhibition of F2RL1 protein were tested.

Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and F2RL1 protein levels were detected by western blot.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish.

The cp-asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing was confirmed via gel electrophoresis.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at which point the asiRNA-containing OPTI-MEM media was replaced with a serum-containing media.

The levels of F2RL1 protein expression were determined via western blot 72 hours after asiRNA transfection. Briefly, the treated A549 cells were lysed with TX-100 lysis buffer (1% TX-100, 150 mM NaCl, 100 mM Tris (pH 8.8)). 10 μg of the total protein extracts were loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Bioworld) and then incubated overnight at 4° C. in 3% BSA containing anti-F2RL1 antibody (Abcam) and anti-GAPDH (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 1×TBST with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL for 1 minute. The F2RL1 and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

The results of the western blot assay are depicted in FIG. 25.

Example 19: Inhibition of F2RL1 mRNA Expression Using Additional F2RL1-Targeting Cp-asiRNAs

A variety of potential cp-asiF2RL1#22 and #50 structures having different strand lengths and number of 2′-O-methylation modifications and phosphorothioate bond were synthesized and tested for their ability to inhibit F2RL1 expression (Table 10).

TABLE 10 Additional cp-asiRNA sequences. SEQ ID NO: 666 F2RL1#50-PS3/19(5, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmAA*mC*A*cholesterol 3′ 667 F2RL1#50-PS3/19(5, 4) (AS): 5′ UGUUUGCCUUCUUCmC*mU*mG*mG*mA 3′ 668 F2RL1#50-PS3/21(7, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmAA*mC*A*cholesterol 3′ 669 F2RL1#50-PS3/21(7, 4) (AS): 5′ UGUUUGCCUUCUUCmCmUmG*mG*mA*mG*mU 3′ 670 F2RL1#50-PS4/19(5, 4) (S): 5′ mAGmGAmAGmAAmGGmCAmA*A*mC*A*cholesterol 3′ 671 F2RL1#50-PS4/19(5, 4) (AS): 5′ UGUUUGCCUUCUUCmC*mU*mG*mG*mA 3′ 672 F2RL1#22-PS3/19(4, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmCU*mG*U*cholesterol 3′ 673 F2RL1#22-PS4/19(4, 4) (AS): 5′ ACAGAGAGGAGGUCmA*mG*mC*mC*A 3′ 674 F2RL1#22-PS3/21(4, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmCU*mG*U*cholesterol 3′ 675 F2RL1#22-PS4/21(4, 4) (AS): 5′ ACAGAGAGGAGGUCmAmGmC*mC*A*A*G 3′ 676 F2RL1#22-P54/19(4, 4) (S): 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ 677 F2RL1#22-P54/19(4, 4) (AS): 5′ ACAGAGAGGAGGUCmA*mG*mC*mC*A 3′ m = 2′-O-Methyl RNA, *= phosphorothioate bond.

The ability of 1 μM and 3 μM of each of the potential cp-asiRNAs listed in Table 10 to inhibit F2RL1 mRNA in A549 cells was tested.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish.

The potential cp-asiRNAs listed in Table 4 were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing was confirmed via gel electrophoresis.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at which point the asiRNA-containing OPTI-MEM media was replaced with a serum-containing media.

The level of F2RL1 mRNA expression was determined 48 hours after asiRNA treatment.

The level of F2RL1 inhibition by 8 cp-asiRNAs is provided in FIG. 26.

Example 20: Inhibition of F2RL1 Protein Expression Using Additional F2RL1-Targeting Cp-asiRNAs

The efficacy of cp-asiRNAs for the inhibition of F2RL1 protein was tested.

Each potential cp-asiRNA was incubated with A549 cells at 1 μM and 3 μM without a delivery reagent and F2RL1 protein levels were measured by western blot.

A549 cells (ATCC) that had been cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco), 100 μg/ml penicillin/streptomycin in a 100 mm cell culture dish.

The cp-asiRNAs were incubated at 95° C. for 5 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing was confirmed via gel electrophoresis.

One day prior to treatment, 2.5×10⁴ A549 cells were seeded in 24-well plates. Immediately before treatment, the A549 cells were washed with Dulbecco's modified Eagle's medium (Gibco) then cultured in the presence of the potential cp-asiRNAs in OPTI-MEM buffer for 24 hours, at which point the asiRNA-containing OPTI-MEM media was replaced with a serum-containing media.

The level of F2RL1 protein expression was determined via western blot 72 hours after asiRNA treatment. Briefly, the treated A549 cells were lysed with TX-100 lysis buffer (1% TX-100, 150 mM NaCl, 100 mM Tris (pH 8.8)). 10 μg of the total protein extracts were loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 V. After electrophoresis, the proteins were transferred to PVDF membrane (Bio-rad) already activated by methanol (Merck) for 1 hour at 300 mA. The membrane was blocked for 1 hour at the room temperature with 3% BSA (Bioworld) and then incubated overnight at 4° C. in 3% BSA containing anti-F2RL1 antibody (Abcam) and anti-GAPDH antibody (Santa Cruz). The membrane was then washed with 1×TBST for 10 minutes three times and was incubated for 1 hour at the room temperature in 1×TBST with HRP-conjugated secondary antibody. The membrane was washed with 1×TBST for 10 minutes and treated with 1×ECL for 1 minute. The F2RL1 and GAPDH bands were then imaged using a Chemidoc instrument (Bio-rad).

The results of the western blot assay are depicted in FIG. 27.

Example 21: In vivo Efficacy Study

After shaving the dorsal region of NC/Nga mice, Dermatophagoides farinae body extract (Df) cream was painted in presented schedule for inducing atopic dermatitis. At day 11, 14 and 18, cp-asiRNA was administered by intradermal injection or spreading of cream emulsified cp-asiRNA before Biostir® AD ointment application (FIG. 29). The dose of intradermal injection was 80 μg/50 μl*4 sites/head, and the dose of the cream emulsified cp-asiRNA was 800 μg/head. Mouse behavior was recorded and scratching behavior for 480 seconds were analyzed. Increased scratching time was observed in Dermatophagoides farinae body extract (DO cream treated samples (1×PBS+Df). In both intradermal injection (FIG. 30, Part A) and cream emulsified cp-asiRNA application (FIG. 30, Part B) condition, IL4RA#5-P53/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-PS4/19 (4,4) treated samples showed reduced scratching time compared to vehicle control (1×PBS+Df). Results are presented as bar graph (mean±S.D) in FIG. 30. The results were statistically analyzed by Student's t-test methods (n=5).

Transepidermal water loss (TEWL) was measured using hand-held evaporimeter (VapoMeter, Delfin Technologies Ltd, Kuopio, Finland). Increased TEWL was observed in Dermatophagoides farinae body extract (Df) cream treated samples (1×PBS+Df). In both intradermal injection (FIG. 31, Part A) and cream emulsified cp-asiRNA application (FIG. 31, Part B) conditions, IL4RA#5-P53/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-PS4/19(4,4) treated samples showed reduced TEWL compared to vehicle control (1×PBS+Df), Data are expressed as mean±S.E.M. The results were statistically analyzed by Student's t-test methods (n=5).

Histological analysis of treated skin region was conducted. Upper panels show H&E staining of skin sections and lower panels show quantified epidermis area by analyzing skin section image. Increased epidermis region thickness, hyperkeratosis, and acanthosis was observed in Dermatophagoides farinae body extract (DO cream treated samples (+Df). In both intradermal injection (FIG. 32, Part A) and cream emulsified cp-asiRNA application (FIG. 32, Part B) condition, IL4RA#5-P53/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-P54/19(4,4) treated samples showed reduced symptoms caused by Df treatment compared to vehicle control (+Df). In both intradermal injection (FIG. 32, Part A) and cream emulsified cp-asiRNA application (FIG. 32, Part B) conditions, IL4RA#5-P53/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-PS4/19 (4,4) treated samples showed reduced level of epidermis thickness compared to vehicle control (1×PBS+Df). Data are expressed as mean±S.E.M. The results were statistically analyzed by student's t-test methods (n=5).

Mast cell infiltration analysis of treated skin region was conducted. FIG. 33 shows toluidine blue staining of skin sections and quantification results of the stained skin section image. Increased mast cell infiltration was observed in Dermatophagoides farinae body extract (Df) cream treated samples (+Df). In both intradermal injection (FIG. 33, Part A) and cream emulsified cp-asiRNA application (FIG. 33, Part B) conditions, IL4RA#5-PS3/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-PS4/19 (4,4) treated samples showed reduced mast cell infiltration compared to vehicle control (+Df). In both intradermal injection and cream emulsified cp-asiRNA application conditions, IL4RA#5-P53/19(4,4), TRPA1#81-PS3/19(4,4), F2RL1#22-PS4/19 (4,4) treated samples showed reduced level of mast cell infiltration area compared to vehicle control (1×PBS+Df). Data are expressed as mean±S.E.M. The results were statistically analyzed by Student's t-test methods (n=5).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

What is claimed is:
 1. An RNA complex comprising an antisense strand of at least 19 nucleotides (nt) in length having sequence complementarity to an F2RL1 mRNA sequence and a sense strand of 15 to 17 nt in length having sequence complementarity to the antisense strand, wherein the antisense strand and the sense strand form a complex in which the 5′ end of the antisense strand and the 3′ end of the sense strand form a blunt end, and wherein the RNA complex has a double-stranded region of 15-17 nt in length and an overhang of at least 2 nt in length formed at the 3′ end of the antisense strand.
 2. The RNA complex of claim 1, wherein the antisense strand is 19 to 21 nt in length.
 3. The RNA complex of claim 1, wherein the antisense strand is at least 24 nt in length.
 4. The RNA complex of claim 1, wherein the sense strand comprises the sequence of SEQ ID NO:
 420. 5. The RNA complex of claim 1, wherein the antisense strand comprises the sequence of SEQ ID NO:
 421. 6. The RNA complex of claim 1, wherein the sense strand comprises the sequence of SEQ ID NO: 420 and the antisense strand comprises the sequence of SEQ ID NO:
 421. 7. The RNA complex of claim 1, wherein the RNA complex comprises a chemical modification.
 8. The RNA complex of claim 7, wherein the chemical modification is a 2′-O-methylated nucleoside, a phosphorothioate bond or a hydrophobic moiety.
 9. The chemical modification of claim 8, wherein the hydrophobic moiety is a cholesterol moiety.
 10. The RNA complex of claim 7, wherein the RNA complex comprises at least one phosphorothioate bond.
 11. The RNA complex of claim 10, wherein the RNA complex is a modified RNA complex selected from: (a) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ (SEQ ID NO: 642); and an antisense strand of: 5′ ACAGAGAGGAGGUCmAmGC*C*A*A*G 3′ (SEQ ID NO: 643); (b) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ (SEQ ID NO: 644); and an antisense strand of: 5′ ACAGAGAGGAGGUCmAmGmC*mC*A*A*G 3′ (SEQ ID NO: 645); (c) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ (SEQ ID NO: 646); and an antisense strand of 5′ ACAGAGAGGAGGUCmAmGmC*mC*mA*mA*mG 3′ (SEQ ID NO: 647); (d) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmCU*mG*U*cholesterol 3′ (SEQ ID NO: 672); and an antisense strand of 5′ ACAGAGAGGAGGUCmA*mG*mC*mC*A 3′ (SEQ ID NO: 673); (e) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmCU*mG*U*cholesterol 3′ (SEQ ID NO: 674); and an antisense strand of 5′ ACAGAGAGGAGGUCmAmGmC*mC*A*A*G 3′ (SEQ ID NO: 675); and (f) an RNA complex consisting of: a sense strand of: 5′ mCUmGAmCCmUCmCUmCUmC*U*mG*U*cholesterol 3′ (SEQ ID NO: 676); and an antisense strand of 5′ ACAGAGAGGAGGUCmA*mG*mC*mC*A 3′ (SEQ ID NO: 677); wherein m represents 2′-O-methyl RNA and * represents a phosphorothioate bond.
 12. The RNA complex of claim 10, wherein the RNA complex is capable of penetrating the cellular membrane of a cell in the absence of a delivery vehicle.
 13. A pharmaceutical composition comprising an RNA complex of claim 1 and a pharmaceutically acceptable carrier.
 14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is formulated for inhalation or for topical administration. 